Method of reverse transcription

ABSTRACT

The present invention relates to reverse transcription of RNA, and in particular to reverse transcription by thermostable DNA polymerases.  Thermoactinomyces vulgaris  and  Bacillus stearothermophilus  possess reverse transcriptase activity in the presence of magnesium or manganese ions. Methods, compositions, and kits for reverse transcription and RT-PCR are also provided.

FIELD OF THE INVENTION

The present invention relates to reverse transcription of RNA, and inparticular to thermostable DNA polymerases that have reversetranscriptase activity.

BACKGROUND

Many ribonucleic acid (RNA) molecules contain secondary structure thatresults from hybridization between complementary regions within the RNAmolecule. A variety of secondary structures can be formed, includinghairpins and cruciforms, RNA molecules containing secondary structureare often difficult to reverse transcribe because polymerases cannotreadily process through the secondary structure.

Because of the difficulty of reverse transcribing RNA molecules withsecondary structure, many techniques dependent upon reversetranscription yield anomalous results. For example, RNA molecules withsecondary structure may be poorly represented in cDNA libraries.Populations of RNA with secondary structure may also yield cDNAlibraries with a short insert size. Furthermore, RNA moleculescontaining secondary structure may be difficult to detect in assays suchas reverse transcription-polymerase chain reaction (RT-PCR).

Traditionally, reverse transcription has been performed with reversetranscriptases encoded by retroviruses (e.g., avian myoblastosis virus(AMV) reverse transcriptase and Moloney murine leukemia virus (MMLV)reverse transcriptase). Several mesophillic DNA polymerases (e.g., E.coli DNA polymerase I) have also been shown to possess reversetranscriptase activity. However, these enzymes are generally used attemperatures of between about 37° C. to 42° C., a temperature rangewhere secondary structure can be a significant problem.

Several thermophilic DNA polymerases (e.g., Thermus aquaticus DNApolymerase and Thermus thermophilus DNA polymerase) also have reversetranscriptase activity. These enzymes are useful for reversetranscription, because at the high temperatures where such enzymes arestable, secondary structure in RNA molecules is reduced. Furthermore,such enzymes can be used to directly synthesize second strand DNA andpotentially even to directly amplify an RNA target. However, the utilityof these thermostable enzymes is limited because they require manganeseas a co-factor for reverse transcriptase activity (e.g., U.S. Pat. No.5,322,770) resulting in deleterious effects. In some cases, the fidelityof the polymerase is reduced as compared to the fidelity of the enzymein the presence of other cofactors, such as magnesium ions. Therefore,it is not desirable to amplify the template in the same reaction mixturein which reverse transcription reaction is conducted. This necessitatesextra time consuming steps when performing RT-PCR. In other cases, thepresence of manganese ions may also cause degradation of the RNAtemplate.

Accordingly, what is needed in the art are alternative thermostablepolymerases that have reverse transcriptase activity. Preferably, suchthermostable polymerases should have reverse transcriptase activity inthe presence of magnesium so that high-fidelity cDNAs may be obtainedand so that both reverse transcription and amplification in RT-PCRreactions may conducted in the same reaction mixture.

SUMMARY OF THE INVENTION

The present invention relates to reverse transcription of RNA templates,and in particular to reverse transcription by thermostable DNApolymerases. The present invention is not limited to any particular RNAtemplate. Indeed, a variety of RNA templates are contemplated. Examplesof RNA templates include, but are not limited to, mRNA, rRNA, purifiedRNA, mixtures of mRNA, mixtures of rRNA and mRNA, and purifiedpreparations of these various RNAs.

The present invention is not limited to the use of a particularthermostable DNA polymerase. Indeed, the use of a variety ofthermostable DNA polymerases is contemplated. In some embodiments, thethermostable DNA polymerase is selected from Thermoactinomyces vulgaris(Tvu) and Bacillus stearothermophilus (Bst) DNA polymerases. In someembodiments, the thermostable DNA polymerase is purified from naturalsources, while in other embodiments, the DNA polymerase is generated byrecombinant techniques. In still other embodiments, the thermostable DNApolymerase lacks significant 5′ exonuclease activity. In someembodiments, the Tvu polymerase is encoded by an amino acid sequenceselected from SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO 6, and variantsor portions thereof. In other embodiments, the Bst polymerase is encodedby an amino acid sequence selected from SEQ ID NO: 19 and SEQ ID NO: 21,and variants or portions thereof.

In some embodiments, the present invention provides methods for reversetranscribing template RNA (i.e., making cDNA copies of the templateRNA). In some embodiments, the method comprises a) providing: i) apolymerase selected from T. vulgaris and B. stearothermophilus DNApolymerases; ii) template RNA; iii) at least one primer; and iv) areaction buffer comprising magnesium ions; b) combining the polymerase,template RNA, at least one primer, and reaction buffer to form areaction mixture; and c) reacting said reaction mixture under conditionssuch that the template RNA is reverse transcribed, producing cDNA. Themethod is not limited by the order in which the polymerase, templateRNA, at least one primer, and reaction buffer are combined. In someembodiments, the reaction buffer is substantially free of manganeseions. In other embodiments, the reacting step is performed at about 50degrees Celsius to about 80 degrees Celsius, preferably at about 60degrees Celsius to about 75 degrees Celsius. The method is not limitedto a particular type of primer. Indeed a variety of primers may be used,including, but not limited to, oligonucleotides complementary to the 5′untranslated region of an mRNA, the coding region of an mRNA, or the 3′untranslated region of an mRNA, oligo(dT), and random primers (e.g.,random hexamers or octamers). In still further embodiments, the methodcomprises the additional step d) amplifying the cDNA produced by thereverse transcription reaction.

The present invention also provides methods for detecting the presenceof an RNA molecule in a sample by reverse transcription PCR (RT-PCR). Insome embodiments, the reverse transcription and amplification reactionsare conducted in the same reaction buffer (i.e., a single pot reactionis performed). In other embodiments, reverse transcription andamplification are performed in separate reactions (i.e., a two potreaction is performed). Accordingly, in some embodiments, the methodcomprises: a) providing: i) a polymerase selected from T. vulgaris andB. stearothermophilus DNA polymerases; ii) a sample suspected ofcontaining a target RNA; iii) at least a first primer and a secondprimer, wherein the first primer is complementary to the target RNA andthe second primer is complementary to a cDNA copy of the target RNA; andiv) a reaction buffer comprising magnesium ions; b) mixing thepolymerase, target RNA, reaction buffers, and primers to form a reactionmixture; c) reacting the reaction mixture under conditions such that thepolymerase reverse transcribes the target RNA to produce first strandDNA; and d) reacting the first strand DNA with the second primer underconditions such that second strand DNA is produced; and e) reacting thefirst strand DNA, second strand DNA, first primer, and second primerunder conditions such that a DNA molecule comprising a third strand anda fourth strand is produced, the third strand having a region ofcomplementarity to the first strand and the fourth strand having aregion of complementarity to the second strand. In some embodiments, thereaction mixture further comprises an additional thermostable polymerase(e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, andthe like). In some embodiments, the conditions further comprise heatingthe reaction mixture. In other embodiments, the conditions furthercomprise cooling the mixture to a temperature at which the thermostableDNA polymerase can conduct primer extension. In still furtherembodiments, the heating and cooling is repeated one or more times.

In still further embodiments, the present invention provides mixturesand kits for performing reverse transcription. In some embodiments, themixture comprises a polymerase selected from T. vulgaris and B.stearothermophilus DNA polymerases, purified RNA, and magnesium ions. Inother embodiments, the concentration of magnesium is from about 0.1 to10 mM, preferably 1 to 5 mM. In another embodiment, the mixture furthercomprises a buffer. In still other embodiments, the mixture comprises asurfactant. In further embodiments, the mixture has a pH of about 6 to10, preferably a pH of about 7 to 9. In some embodiments, the reactionmixture further comprises an additional thermostable polymerase (e.g.,Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and thelike)

In other embodiments of the present invention, a kit is provided. Insome embodiments, the kit comprises a polymerase selected from T.vulgaris and B. stearothermophilus DNA polymerases, purified RNA as acontrol template, and a buffer comprising magnesium ions. In someembodiments, the kit contains instructions for performing reversetranscription. In other embodiments, the buffer further comprises asurfactant. In some embodiments, the pH of the buffer is from about 6 to10, preferably a pH of about 7 to 9. In some embodiments, the kitcomprises an additional thermostable polymerase (e.g., Taq DNApolymerase, Tne DNA polymerase, Pfu DNA polymerase, and the like)

The present invention also provides methods for amplifying a doublestranded DNA molecule, comprising the steps of: a) providing: i) a firstDNA molecule comprising a first strand and a second strand, wherein thefirst and second strands are complementary to one another; ii) a firstprimer and a second primer, wherein the first primer is complementary tothe first DNA strand, and the second primer is complementary to thesecond DNA strand; and iii) a first thermostable DNA polymerase derivedfrom T. vulgaris; and b) mixing the first DNA molecule, first primer,second primer, and polymerase to form a reaction mixture underconditions such that a second DNA molecule comprising a third strand anda fourth strand are synthesized, with the third strand having a regioncomplementary to the first strand and the fourth strand having a regioncomplementary to the second strand. In some embodiments, the reactionmixture further comprises an additional thermostable polymerase (e.g.,Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and thelike). The method of the present invention is not limited by the sourceof the first DNA molecule. In a preferred embodiment, the first DNAmolecule is present in a genomic DNA mixture (e.g., in genomic DNAextracted from an organism, tissue or cell line). In alternativeembodiments, the first DNA molecule is derived from an RNA moleculeusing reverse transcriptase-PCR (RT-PCR). The newly synthesized DNAmolecule (cDNA) then serves as substrate in the subsequent amplificationreaction. The conditions that permit the primer to hybridize to the DNAmolecule, and allow the DNA polymerase to conduct primer extension maycomprise the use of a buffer.

In one embodiment, the method comprises heating the mixture. In analternative embodiment, the method further comprises cooling the mixtureto a temperature at which the thermostable DNA polymerase can conductprimer extension. In a particularly preferred embodiment, the methodcomprises repeating the heating and cooling steps one or more times.

It is also contemplated that the polymerase of the method will havevarious useful properties. It is therefore contemplated that in oneembodiment of the method, the Tvu polymerase lacks significant 5′-3′exonuclease activity. In other embodiments, the polymerase has reversetranscriptase activity in the presence of either magnesium or manganeseions. In still other embodiments, the reverse transcriptase activity inpresence of magnesium ions is substantially manganese ion independent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the single letter code nucleotide for the DNA sequenceencoding full-length Tvu DNA polymerase (SEQ ID NO: 1).

FIG. 2 provides the predicted amino acid sequence of full-length Tvu DNApolymerase (SEQ ID NO: 2).

FIG. 3 provides the DNA sequence encoding the 5′ to 3′ exonucleasedeletion mutant form of Tvu DNA polymerase called M285. This DNAsequence encodes the enzyme beginning at the nucleotides encoding themethionine amino acid at position 285 of wild type Tvu DNA polymeraseand ending at the termination codon of the wild type enzyme (SEQ ID NO:3).

FIG. 4 provides the predicted amino acid sequence of M285 Tvu DNApolymerase (SEQ ID NO: 4).

FIG. 5 provides the DNA sequence encoding the 5′ to 3′ exonucleasedeletion mutant form of Tvu DNA polymerase called T289M. This DNAsequence encodes the enzyme beginning at amino acid 289 of the wild typeTvu DNA polymerase, mutated to encode a methionine instead of threoninethat appears at this position in wild type, and ending at thetermination codon of the wild type enzyme (SEQ ID NO: 5).

FIG. 6 provides the predicted amino acid sequence of T289M Tvu DNApolymerase (SEQ ID NO: 6).

FIG. 7 provides the complete coding sequence for Bacillusstearothermophilus DNA polymerase 1, Genbank Accession No. U33536 (SEQID NO: 18).

FIG. 8 provides the amino acid sequence (SEQ ID NO: 19) encoded by SEQID NO. 18.

FIG. 9 provides the coding sequence for Bacillus stearothermophilus DNApolymerase 1 lacking 5′ to 3′ exonuclease activity, Genbank AccessionNo.: AR053713 (SEQ ID NO: 20).

FIG. 10 provides the amino acid sequence (SEQ ID NO: 21, GenbankAccession No. AAE15301) encoded by SEQ ID NO: 20.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term “gene” as used herein, refers to a DNA sequence that comprisescontrol and coding sequences necessary for the production of apolypeptide or protein precursor. The polypeptide can be encoded by afull length coding sequence or by any portion of the coding sequence, aslong as the desired protein activity is retained.

“Nucleoside”, as used herein, refers to a compound consisting of apurine [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine(U), or cytidine (C)] base covalently linked to a pentose, whereas“nucleotide” refers to a nucleoside phosphorylated at one of its pentosehydroxyl groups.

A “nucleic acid”, as used herein, is a covalently linked sequence ofnucleotides in which the 3′ position of the pentose of one nucleotide isjoined by a phosphodiester group to the 5′ position of the pentose ofthe next, and in which the nucleotide residues (bases) are linked inspecific sequence; i.e., a linear order of nucleotides. A“polynucleotide”, as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. An“oligonucleotide”, as used herein, is a short polynucleotide or aportion of a polynucleotide. An oligonucleotide typically contains asequence of about two to about one hundred bases. The word “oligo” issometimes used in place of the word “oligonucleotide”.

Nucleic acid molecules are said to have a “5′-terminus” (5′ end) and a“3′-terminus” (3′ end) because nucleic acid phosphodiester linkagesoccur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a polynucleotide at which a newlinkage would be to a 5′ carbon is its 5′ terminal nucleotide. The endof a polynucleotide at which a new linkage would be to a 3′ carbon isits 3′ terminal nucleotide. A terminal nucleotide, as used herein, isthe nucleotide at the end position of the 3′- or 5′-terminus.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring.

As used herein, a nucleic acid sequence, even if internal to a largeroligonucleotide or polynucleotide, also may be said to have 5′ and 3′ends. In either a linear or circular DNA molecule, discrete elements arereferred to as being “upstream” or 5′ of the “downstream” or 3′elements. This terminology reflects the fact that transcription proceedsin a 5′ to 3′ fashion along the DNA strand. Typically, promoter andenhancer elements that direct transcription of a linked gene aregenerally located 5′ or upstream of the coding region. However, enhancerelements can exert their effect even when located 3′ of the promoterelement and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

Polypeptide molecules are said to have an “amino terminus” (N-terminus)and a “carboxy terminus” (C-terminus) because peptide linkages occurbetween the backbone amino group of a first amino acid residue and thebackbone carboxyl group of a second amino acid residue. Typically, theterminus of a polypeptide at which a new linkage would be to thecarboxy-terminus of the growing polypeptide chain, and polypeptidesequences are written from left to right beginning at the aminoterminus.

The term “wild-type” as used herein, refers to a gene or gene productthat has the characteristics of that gene or gene product isolated froma naturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “wild-type” form of the gene. In contrast, the term “mutant” refersto a gene or gene product that displays modifications in sequence and/orfunctional properties (i.e., altered characteristics) when compared tothe wild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct. The wild-type form of the coding region for the Tvu DNApolymerase is shown in SEQ ID NO: 1; the wild-type form of the Tvu DNApolymerase protein is shown in SEQ ID NO: 2. Tvu DNA polymerase proteinsencoded by “mutant” genes are referred to as “variant” Tvu DNApolymerases. Tvu DNA polymerase proteins encoded by “modified” or“mutant” genes are referred to as “non-naturally occurring” or “variant”Tvu DNA polymerases. Tvu DNA polymerase proteins encoded by thewild-type Tvu DNA polymerase gene (i.e., SEQ ID NO:1) are referred to as“naturally occurring” Tvu DNA polymerases.

As used herein, the term “sample template” refers to a nucleic acidoriginating from a sample which is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template, which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids other than those to be detected may bepresent as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally (e.g., as in a purified restriction digest) orproduced synthetically, which is capable of acting as a point ofinitiation of nucleic acid synthesis when placed under conditions inwhich synthesis of a primer extension product which is complementary toa nucleic acid strand is induced (i.e., in the presence of nucleotides,an inducing agent such as DNA polymerase, and under suitable conditionsof temperature and pH). The primer is preferably single-stranded formaximum efficiency in amplification, but may alternatively bedouble-stranded. If double-stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and useof the method.

A primer is said to be “capable of hybridizing to a DNA molecule” ifthat primer is capable of annealing to the DNA molecule; that is theprimer shares a degree of complementarity with the DNA molecule. Thedegree of complementarity can be, but need not be, complete (i.e., theprimer need not be 100% complementary to the DNA molecule). Indeed, whenmutagenic PCR is to be conducted, the primer will contain at least onemismatched base which cannot hybridize to the DNA molecule. Any primerwhich can anneal to and support primer extension along a template DNAmolecule under the reaction conditions employed is capable ofhybridizing to a DNA molecule.

As used herein, the terms “complementary” or “complementarity” are usedin reference to a sequence of nucleotides related by the base-pairingrules. For example, for the sequence 5′ “A-G-T” 3′, is complementary tothe sequence 3′ “T-C-A” 5′. Complementarity may be “partial,” in whichonly some of the nucleic acids' bases are matched according to the basepairing rules. Or, there may be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as detection methodswhich depend upon hybridization of nucleic acids.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally (e.g., as in apurified restriction digest) or produced synthetically, recombinantly orby PCR amplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that theprobe used in the present invention is labeled with any “reportermolecule,” so that it is detectable in a detection system, including,but not limited to enzyme (i.e., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label. The terms “reporter molecule” and“label” are used herein interchangeably. In addition to probes, primersand deoxynucleoside triphosphates may contain labels; these labels maycomprise, but are not limited to, ³²P, ³³P, ³⁵S, enzymes, or fluorescentmolecules (e.g., fluorescent dyes).

As used herein, the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid ofinterest bounded by the primers. In PCR, this is the region amplifiedand/or identified. Thus, the “target” is sought to be isolated fromother nucleic acid sequences. A “segment” is defined as a region ofnucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method described in U.S. Pat. Nos. 4,683,195, 4,889,818, and4,683,202, all of which are hereby incorporated by reference. Thesepatents describe methods for increasing the concentration of a segmentof a target sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase (e.g.,Taq). The two primers are complementary to their respective strands ofthe double stranded target sequence. To effect amplification, themixture is denatured and the primers then annealed to theircomplementary sequences within the target molecule. Following annealing,the primers are extended with a polymerase so as to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing andpolymerase extension can be repeated many times (i.e., denaturation,annealing and extension constitute one “cycle”; there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(hereinafter “PCR”). Because the desired amplified segments of thetarget sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (i.e., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR process itself are, themselves, efficient templates for subsequentPCR amplifications.

As used herein, the terms “PCR product” and “PCR fragment” refer to theresultant mixture of compounds after two or more cycles of the PCR stepsof denaturation, annealing and extension are complete. These termsencompass the case where there has been amplification of one or moresegments of one or more target sequences.

A DNA polymerase is said herein to be “derived from the eubacterium T.vulgaris” if that polymerase comprises all or a portion of the aminoacid sequence of the Tvu DNA polymerase of SEQ ID NO: 2 and maintainsDNA synthesis activity. DNA polymerases derived from T. vulgaris includethe native Tvu DNA polymerase isolated from T. vulgaris cells, as wellas recombinant Tvu DNA polymerases encoded by the wild-type Tvu DNApolymerase gene (SEQ ID NO: 1) or mutant or variants thereof whichmaintain DNA synthesis activity (including those containing amino acidanalogs).

The term “full-length thermostable Tvu DNA polymerase” as used herein,refers to a DNA polymerase that encompasses essentially every amino acidencoded by the Tvu DNA polymerase gene (SEQ ID NO: 1). One skilled inthe art knows there are subtle modifications of some proteins in livingcells so that the protein is actually a group of closely relatedproteins with slight alterations. For example, some but not allproteins: a) have amino acids removed from the amino-terminus; and/or b)have added chemical groups (e.g., glycosylation groups). Thesemodifications may result in molecular weight increase or decrease. Thesetypes of modifications are typically heterogenous. Thus, not allmodifications occur in every molecule. Thus, the natural “full-length”molecule may actually be a family of molecules that start from the sameamino acid sequence but have small differences in their modifications.The term “full-length thermostable Tvu DNA polymerase” encompasses sucha family of molecules. The Tvu DNA polymerase gene encodes a protein of876 amino acids having a predicted molecular weight of 96.3 kilodaltons(kD). As shown in the examples below, the full-length polymerasemigrates with an apparent molecular weight of about 97 kD on a 4-20%gradient Tris-glycine PAGE.

The term “high fidelity polymerase” refers to DNA polymerases with errorrates of 5×10⁻⁶ per base pair or lower. Examples of high fidelity DNApolymerases include the Tli DNA polymerase derived from Thermococcuslitoralis (Promega, NEB), Pfu DNA polymerase derived from Pyrococcusfuriosus (Stratagene), and Pwo DNA polymerase derived from Pyrococcuswoesii (BM). The error rate of a DNA polymerase may be measured usingassays known to the art.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule expressed from a recombinant DNAmolecule. In contrast, the term “native protein” is used herein toindicate a protein isolated from a naturally occurring (i.e., anonrecombinant) source. Molecular biological techniques may be used toproduce a recombinant form of a protein with identical properties ascompared to the native form of the protein. The term “rTvu” is used todesignate a recombinant form of Tvu polymerase. The term “nTvu” is usedto designate the native form of Tvu polymerase. Tvu polymeraseencompasses both nTvu and rTvu polymerase.

As used herein in reference to an amino acid sequence or a protein, theterm “portion” (as in “a portion of an amino acid sequence”) refers tofragments of that protein. The fragments may range in size from fouramino acid residues to the entire amino acid sequence minus one aminoacid. When used in relation to Tvu polymerases, the fragments may rangein size from greater than or equal to about 300 amino acid residues,more preferably greater than or equal to about 400 amino acid residues,most preferably greater to or equal to about 500 amino acids to theentire amino acid sequence minus one amino acid. Particularly preferredfragments of Tvu polymerases retain one or more of the enzymaticactivities associated with the wild-type Tvu polymerase (i.e., 5′exonuclease, 3′ exonuclease and polymerization activity)

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (e.g., Tvu DNA polymerases andfragments thereof) joined to an exogenous protein fragment (e.g., thefusion partner which consists of a non-Tvu polymerase protein). Thefusion partner may enhance the solubility of Tvu polymerase protein asexpressed in a host cell, may provide an affinity tag to allowpurification of the recombinant fusion protein from the host cell orculture supernatant, or both. If desired, the fusion protein may beremoved from the protein of interest (e.g., Tvu DNA polymerase orfragments thereof) by a variety of enzymatic or chemical means know tothe art.

The term “5′ to 3′ exonuclease activity” refers to the presence of anactivity in a protein that is capable of removing nucleotides from the5′ end of an oligonucleotide. 5′ to 3′ exonuclease activity may bemeasured using any of the assays provided herein or known in the art.The term “substantially free of 5′ to 3′ exonuclease activity” indicatesthat the protein has less than about 5% of the 5′ to 3′ exonucleaseactivity of wild-type Tvu, preferably less than about 3% of the 5′ to 3′exonuclease activity of wild-type Tvu, and most preferably no detectable5′ to 3′ exonuclease activity.

The term “3′ to 5′ exonuclease activity” refers to the presence of anactivity in a protein that is capable of removing nucleotides from the3′ end of an oligonucleotide. The 3′ to 5′ exonuclease activity may bemeasured using any of the assays provided herein or known in the art.The term “substantially free of 3′ to 5′ exonuclease activity” indicatesthat the protein has less than about 5% of the 3′ to 5′ exonucleaseactivity of wild-type Tvu, preferably less than about 3% of the 3′ to 5′exonuclease activity of wild-type Tvu, and most preferably no detectable3′ to 5′ exonuclease activity.

The terms “DNA polymerase activity,” “synthesis activity” and“polymerase activity” are used interchangeably and refer to the abilityof a DNA polymerase to synthesize new DNA strands by the incorporationof deoxynucleoside triphosphates. The examples below provide assays forthe measurement of DNA polymerase activity, although a number of suchassays are known in the art. A protein capable of directing thesynthesis of new DNA strands by the incorporation of deoxynucleosidetriphosphates in a template-dependent manner is said to be “capable ofDNA synthesis activity.”

The term “reduced levels of 5′ to 3′ exonuclease” is used in referenceto the level of 5′ to 3′ exonuclease activity displayed by the wild-typeTvu DNA polymerase (i.e., the polymerase of SEQ ID NO:2) and indicatesthat the mutant polymerase exhibits lower levels of 5′ to 3′ exonucleasethan does the full-length or unmodified enzyme, preferably less thanabout 3% of the 5′ to 3′ exonuclease activity of the full-length orunmodified enzyme, and most preferably no detectable 5′ to 3′exonuclease activity.

A polymerase which “lacks significant 5′ to 3′ exonuclease” is apolymerase which exhibits less than about 5% of the 5′ to 3′ exonucleaseactivity of wild-type polymerases, preferably less than about 3% of the5′ to 3′ exonuclease activity of the wild-type enzyme, and mostpreferably no detectable 5′ to 3′ exonuclease activity.

The term “reverse transcriptase activity” and “reverse transcription”refers to the ability of an enzyme to synthesize a DNA strand (i.e.,complementary DNA, cDNA) utilizing an RNA strand as a template. The term“substantially manganese ion independent,” when used in reference toreverse transcriptase activity, refers to reverse transcriptase activityin a reaction mix that contains a low proportion (i.e., less than about5% of the concentration) of manganese compared to magnesium.

The terms “cell,” “cell line,” “host cell,” as used herein, are usedinterchangeably, and all such designations include progeny or potentialprogeny of these designations. The words “transformants” or “transformedcells” include the primary transformed cells derived from that cellwithout regard to the number of transfers. All progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Nonetheless, mutant progeny that have the same functionalityas screened for in the originally transformed cell are included in thedefinition of transformants.

The present invention provides Tvu polymerases expressed in eitherprokaryotic or eukaryotic host cells. Nucleic acid encoding the Tvupolymerase may be introduced into bacterial host cells by a number ofmeans including transformation of bacterial cells made competent fortransformation by treatment with calcium chloride or by electroporation.In embodiments in which Tvu polymerases are to be expressed in the hostcells, nucleic acid encoding the Tvu polymerase may be introduced intoeukaryotic host cells by any suitable means, including calcium phosphateco-precipitation, spheroplast fusion, electroporation and the like. Whenthe eukaryotic host cell is a yeast cell, transformation may beaccomplished by such methods as treatment of the host cells with lithiumacetate or by electroporation.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

Nucleic acids are known to contain different types of mutations. A“point” mutation refers to an alteration in the sequence of a nucleotideat a single base position from the wild type sequence. Mutations mayalso refer to insertion or deletion of one or more bases, so that thenucleic acid sequence differs from the wild-type sequence.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or probe will compete for and inhibit the binding(i.e., the hybridization) of a completely homologous to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target whichlacks even a partial degree of complementarity (e.g., less than about30% identity). In this case, in the absence of non-specific binding, theprobe will not hybridize to the second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or a genomic clone, the term “substantially homologous” refersto any probe which can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described herein.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acid strands. Hybridization and thestrength of hybridization (i.e., the strength of the association betweennucleic acid strands) is impacted by many factors well known in the artincluding the degree of complementarity between the nucleic acids,stringency of the conditions involved affected by such conditions as theconcentration of salts, the T_(m) (melting temperature) of the formedhybrid, the presence of other components (e.g. the presence or absenceof polyethylene glycol), the molarity of the hybridizing strands and theG:C content of the nucleic acid strands.

As used herein, the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. With“high stringency” conditions, nucleic acid base pairing will occur onlybetween nucleic acid fragments that have a high frequency ofcomplementary base sequences. Thus, conditions of “medium” or “low”stringency are often required when it is desired that nucleic acidswhich are not completely complementary to one another be hybridized orannealed together. The art knows well that numerous equivalentconditions can be employed to comprise medium or low stringencyconditions. The choice of hybridization conditions is generally evidentto one skilled in the art and is usually guided by the purpose of thehybridization, the type of hybridization (DNA-DNA or DNA-RNA), and thelevel of desired relatedness between the sequences (e.g., Sambrook etal., 1989. Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington D.C., 1985, for a general discussion of the methods).

The stability of nucleic acid duplexes is known to decrease with anincreased number of mismatched bases, and further to be decreased to agreater or lesser degree depending on the relative positions ofmismatches in the hybrid duplexes. Thus, the stringency of hybridizationcan be used to maximize or minimize stability of such duplexes.Hybridization stringency can be altered by: adjusting the temperature ofhybridization; adjusting the percentage of helix destabilizing agents,such as formamide, in the hybridization mix; and adjusting thetemperature and/or salt concentration of the wash solutions. For filterhybridizations, the final stringency of hybridizations often isdetermined by the salt concentration and/or temperature used for thepost-hybridization washes.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature”. The melting temperature is the temperature at which 50% ofa population of double-stranded nucleic acid molecules becomesdissociated into single strands. The equation for calculating the Tm ofnucleic acids is well-known in the art. The T_(m) of a hybrid nucleicacid is often estimated using a formula adopted from hybridizationassays in 1 M salt, and commonly used for calculating T_(m) for PCRprimers: [(number of A+T)×2° C.+(number of G+C)×4° C.]. (C. R. Newton etal., PCR, 2nd Ed., Springer-Verlag (New York, 1997), p. 24). Thisformula was found to be inaccurate for primers longer than 20nucleotides. (Id.) Another simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl. (e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization, 1985).Other more sophisticated computations exist in the art which takestructural as well as sequence characteristics into account for thecalculation of T_(m). A calculated T_(m) is merely an estimate; theoptimum temperature is commonly determined empirically.

The term “isolated” when used in relation to a nucleic acid, as in“isolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant with which it is ordinarily associated in its source. Thus,an isolated nucleic acid is present in a form or setting that isdifferent from that in which it is found in nature. In contrast,non-isolated nucleic acids (e.g., DNA and RNA) are found in the statethey exist in nature. For example, a given DNA sequence (e.g., a gene)is found on the host cell chromosome in proximity to neighboring genes;RNA sequences (e.g., a specific mRNA sequence encoding a specificprotein), are found in the cell as a mixture with numerous other mRNAswhich encode a multitude of proteins. However, isolated nucleic acidencoding a Tvu polymerase includes, by way of example, such nucleic acidin cells ordinarily expressing a Tvu polymerase where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid or oligonucleotide may be presentin single-stranded or double-stranded form. When an isolated nucleicacid or oligonucleotide is to be utilized to express a protein, theoligonucleotide contains at a minimum, the sense or coding strand (i.e.,the oligonucleotide may single-stranded), but may contain both the senseand anti-sense strands (i.e., the oligonucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” means the result ofany process that removes some of a contaminant from the component ofinterest, such as a protein or nucleic acid. The percent of a purifiedcomponent is thereby increased in the sample.

The term “operably linked” as used herein refer to the linkage ofnucleic acid sequences in such a manner that a nucleic acid moleculecapable of directing the transcription of a given gene and/or thesynthesis of a desired protein molecule is produced. The term alsorefers to the linkage of sequences encoding amino acids in such a mannerthat a functional protein is produced.

As used herein, the term “promoter” means a recognition site on a DNAsequence or group of DNA sequences that provide an expression controlelement for a gene and to which RNA polymerase specifically binds andinitiates RNA synthesis (transcription) of that gene.

As used herein, the term “recombinant DNA molecule” means a hybrid DNAsequence comprising at least two nucleotide sequences not normally foundtogether in nature.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another andcapable of replication in a cell. Vectors may include plasmids,bacteriophages, viruses, cosmids, and the like.

The terms “recombinant vector” and “expression vector” as used hereinrefer to DNA or RNA sequences containing a desired coding sequence andappropriate DNA or RNA sequences necessary for the expression of theoperably linked coding sequence in a particular host organism.Prokaryotic expression vectors include a promoter, a ribosome bindingsite, an origin of replication for autonomous replication in host cellsand possibly other sequences, e.g. an optional operator sequence. Apromoter is defined as a DNA sequence that directs RNA polymerase tobind to DNA and to initiate RNA synthesis. Eukaryotic expression vectorsinclude a promoter, polyadenlyation signal and optionally an enhancersequence.

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. Typically, the coding region is bounded on the 5′side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by a stop codon (e.g., TAA, TAG, TGA). Insome cases the coding region is also known to initiate by a nucleotidetriplet “TTG”.

As used herein, the term “a polynucleotide having a nucleotide sequenceencoding a gene,” means a nucleic acid sequence comprising the codingregion of a gene, or in other words the nucleic acid sequence whichencodes a gene product. The coding region may be present in either acDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. In further embodiments, the coding regionmay contain a combination of both endogenous and exogenous controlelements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequence(s). For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al., Science 236:1237, 1987). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells. Promoterand enhancer elements have also been isolated from viruses and analogouscontrol elements, such as promoters, are also found in prokaryotes. Theselection of a particular promoter and enhancer depends on the cell typeused to express the protein of interest. Some eukaryotic promoters andenhancers have a broad host range while others are functional in alimited subset of cell types (for review, see Voss, et al., TrendsBiochem. Sci., 11:287, 1986; and Maniatis, et al., supra 1987). Forexample, the SV40 early gene enhancer is very active in a wide varietyof cell types from many mammalian species and has been widely used forthe expression of proteins in mammalian cells (Dijkema et al., EMBO J.4:761, 1985). Two other examples of promoter/enhancer elements active ina broad range of mammalian cell types are those from the humanelongation factor 1α gene (Uetsuki et al., J. Biol. Chem., 264:5791,1989; Kim, et al., Gene 91:217, 1990; and Mizushima and Nagata, Nuc.Acids. Res., 18:5322, 1990) and the long terminal repeats of the Roussarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777, 1982);and the human cytomegalovirus (Boshart, et al., Cell 41:521, 1985).

As used herein, the term “promoter/enhancer” denotes a segment of DNAcontaining sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element as described above). For example, the long terminalrepeats of retroviruses contain both promoter and enhancer functions.The enhancer/promoter may be “endogenous” or “exogenous” or“heterologous.” An “endogenous” enhancer/promoter is one that isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer/promoter is one that is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques) such that transcription of the gene is directed by thelinked enhancer/promoter.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript ineukaryotic host cells. Splicing signals mediate the removal of intronsfrom the primary RNA transcript and consist of a splice donor andacceptor site (Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press, New York, 1989, pp.16.7-16.8). A commonly used splice donor and acceptor site is the splicejunction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires expression of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly(A) site” or“poly(A) sequence” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable, astranscripts lacking a poly(A) tail are unstable and are rapidlydegraded. The poly(A) signal utilized in an expression vector may be“heterologous” or “endogenous.” An endogenous poly(A) signal is one thatis found naturally at the 3′ end of the coding region of a given gene inthe genome. A heterologous poly(A) signal is one which has been isolatedfrom one gene and positioned 3′ to another gene. A commonly usedheterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A)signal is contained on a 237 bp BamHI/BclI restriction fragment anddirects both termination and polyadenylation (Sambrook, supra, at16.6-16.7).

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequenceswhich allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Vectors containingeither the SV40 or polyoma virus origin of replication replicate to highcopy number (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen. In contrast, vectors containing thereplicons from bovine papillomavirus or Epstein-Barr virus replicateextrachromosomally at low copy number (˜100 copies/cell).

As used herein, the term “enzyme” refers to molecules or moleculeaggregates that are responsible for catalyzing chemical and biologicalreactions. Such molecules are typically proteins, but can also compriseshort peptides, RNAs, ribozymes, antibodies, and other molecules. Amolecule that catalyzes chemical and biological reactions is referred toas “having enzyme activity” or “having catalytic activity.”

As used herein, the term “polymerase” refers to an enzyme thatsynthesizes nucleic acid strands (e.g., RNA or DNA) from ribonucleosidetriphosphates to deoxyribonucleoside triphosphates.

As used herein, the term “polymerase activity” refers to the ability ofan enzyme to synthesize nucleic acid stands (e.g., RNA or DNA) fromribonucleoside triphosphates or deoxynucleoside triphosphates. DNApolymerases synthesize DNA, while RNA polymerases synthesize RNA.

As used herein, the term “surfactant” refers to any molecule having botha polar head group, that energetically prefers solvation by water, and ahydrophobic tail that is not well solvated by water. The term “cationicsurfactant” refers to a surfactant with a cationic head group. The term“anionic surfactant” refers to a surfactant with an anionic head group.

As used herein, the terms “buffer” or “buffering agents” refer tomaterials that when added to a solution, cause the solution to resistchanges in pH.

As used herein, the terms “reducing agent” and “electron donor” refer toa material that donates electrons to a second material to reduce theoxidation state of one or more of the second material's atoms.

The term “monovalent salt” refers to any salt in which the metal (e.g.,Na, K, or Li) has a net 1+ charge in solution (i.e., one more protonthan electron).

As used herein, the term “divalent salt” refers to any salt in which ametal (e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.

As used herein, the terms “chelator” or “chelating agent” refer to anymaterials having more than one atom with a lone pair of electrons thatare available to bond to a metal ion.

As used herein, the term “solution” refers to an aqueous or non-aqueousmixture.

As used herein, the term “buffering solution” refers to a solutioncontaining a buffering reagent.

As used herein, the term “reaction buffer” refers to a bufferingsolution in which an enzymatic reaction is performed.

As used herein, the term “storage buffer” refers to a buffering solutionin which an enzyme is stored.

As used herein, the phrase “substantially free of manganese ions” refersto a solution that is characterized by absence of more than traceamounts of manganese. In functional terms, a solution that is“substantially free of manganese ions” can contain small or traceamounts of manganese ions so that the fidelity of DNA polymerases knownto be sensitive to manganese (e.g., Taq DNA polymerase) is not decreased(e.g., the fidelity is the same as compared to the fidelity of the DNApolymerase in a solution completely free of manganese).

All amino acid residues identified herein are in the naturalL-configuration. In keeping with standard polypeptide nomenclature, J.Biol. Chem., 243:3557-3559, 1969, abbreviations for amino acid residuesare as shown in the following Table of Correspondence.

TABLE OF CORRESPONDENCE 1-Letter 3-Letter AMINO ACID Y Tyr L-tyrosine GGly glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine SSer L-serine I Ile L-isoleucine L Leu L-leucine T Thr L-threonine V ValL-valine P Pro L-proline K Lys L-lysine H His L-histidine Q GlnL-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine DAsp L-aspartic acid N Asn L-asparagine C Cys L-cysteine

DESCRIPTION OF THE INVENTION

The present invention relates to reverse transcription of RNA templates,and in particular to reverse transcription by thermostable DNApolymerases. Extensive research has been conducted on the isolation ofDNA polymerases from mesophilic organisms such as E. coli. (e.g.,Bessman et al., J. Biol. Chem. 223:171, 1957; Buttin and Kornberg, J.Biol. Chem. 241:5419, 1966; and Joyce and Steitz, Trends Biochem. Sci.,12:288-292, 1987). Other mesophilic polymerases have also been studied,including, but not limited to Bacillus licheniformis (Stenesh andMcGowan, Biochim. Biophys. Acta 475:32-44, 1977; and Stenesh and Roe,Biochim. Biophys. Acta 272:156-166, 1.972); Bacillus subtilis (Low etal., J. Biol. Chem., 251:1311, 1976; and Ott et al., J. Bacteriol.,165:951, 1986); Salmonella typhimurium (Harwood et al., J. Biol. Chem.,245:5614, 1970; and Hamilton and Grossman, Biochem., 13:1885, 1974),Streptococcus pneumoniae (Lopez et al., J. Biol. Chem., 264:4255, 1989);and Micrococcus luteus (Engler and Bessman, Cold Spring Harbor Symp.,43:929, 1979).

Somewhat less investigation has been made on the isolation andpurification of DNA polymerases from thermophilic organisms. However,native (i.e., non-recombinant) and/or recombinant thermostable DNApolymerases have been purified from various organisms, as shown in Table1 below. In addition to native forms, modified forms of thermostable DNApolymerases having reduced or absent 5′ to 3′ exonuclease activity havebeen expressed and purified from Thermus aquaticus, T. maritima, Thermusspecies sps17, Thermus species Z05, T. thermophilus, B.stearothermophilus (U.S. Pat. Nos. 5,747,298, 5,834,253, 5,874,282, and5,830,714) and T. africanus (WO 92/06200).

Reverse transcription from many RNA templates by commonly used reversetranscriptases such as avian myeloblastosis virus (AMV) reversetranscriptase and Moloney murine leukemia virus (MMLV) reversetranscriptase is often limited by the secondary structure of the RNAtemplate. Secondary structure in RNA results from hybridization betweencomplementary regions within a given RNA molecule. Secondary structurecauses poor synthesis of cDNA and premature termination of cDNA productsbecause polymerases are unable to process through the secondarystructure. Therefore, RNAs with secondary structure may be poorlyrepresented in a cDNA library and detection of the presence of RNA withsecondary structure in a sample by RT-PCR may be difficult. Furthermore,secondary structure in RNA may cause inconsistent results in techniquessuch as differential display PCR. Accordingly, it is advantageous toconduct reverse transcription reactions at increased temperatures sothat secondary structure is removed or limited.

Several thermostable DNA polymerases (e.g., T. thermophilus DNApolymerase, T. aquaticus DNA polymerase [e.g., U.S. Pat. No. 5,322,170],and A. thermophilum DNA polymerase [e.g., WO 98/14588]) have beendemonstrated to possess reverse transcriptase activity (See Table 1 fora listing of thermostable DNA polymerases).

TABLE 1 Polymerases Isolated From Thermophilic Organisms OrganismCitation Thermus aquaticus Kaledin et al., Biochem., 45: 494-501 (1980);Biokhimiya 45: 644-651 (1980). Chien et al., J. Bacteriol., 127: 1550(1976). University of Cincinnati Master's thesis by A. Chien,“Purification and Characterization of DNA Polymerase from Thermusaquaticus,” (1976). University of Cincinnati, Master's thesis by D. B.Edgar, “DNA Polymerase From an Extreme Thermophile: Thermus aquaticus.”(1974). U.S. Pat. No. 4,889,818* U.S. Pat. No. 5,352,600* U.S. Pat. No.5,079,352* European Patent Pub. No. 258,017* PCT Pub. No. WO 94/26766*PCT Pub. No. WO 92/06188* PCT Pub. No. WO 89/06691* Thermatoga maritimaPCT Pub. No. WO 92/03556* Thermatoga neapolitana U.S. Pat. No.5,912,155* U.S. Pat. No. 5,939,301* U.S. Pat. No. 6,001,645* Thermotogastrain FjSS3-B.1 Simpson et al., Biochem. Cell Biol., 68: 1292-1296(1990). Thermosipho africanus PCT Pub. No. 92/06200* U.S. Pat. No.5,968,799* Thermus thermophilus Myers and Gelfand, Biochem., 30: 7661(1991). PCT Pub. No. WO 91/09950* PCT Pub. No. WO 91/09944* Bechterevaet al., Nucleic Acids Res., 17: 10507 (1989). Glukhov et al., Mol. Cell.Probes 4: 435-443 (1990). Carballeira et al., BioTech., 9: 276-281(1990). Ruttiman et al., Eur. J. Biochem., 149: 41-46 (1985). Oshima etal., J. Biochem., 75: 179-183 (1974). Sakaguchi and Yajima, Fed. Proc.,33: 1492 (1974) (abstract). Thermus flavus Kaledin et al., Biochem., 46:1247-1254 (1981); Biokhimiya 46: 1576-1584 (1981). PCT Pub. No. WO94/26766* Thermus ruber Kaledin et al., Biochem., 47: 1515-1521 (1982);Biokhimiya 47: 1785-1791 (1982). Thermoplasma acidophilum Hamal et al.,Eur. J. Biochem., 190: 517-521 (1990). Forterre et al., Can. J.Microbiol., 35: 228-233 (1989). Sulfolobus acidocaldarius Salhi et al.,J. Mol. Biol., 209: 635-641 (1989). Salhi et al., Biochem. Biophys. Res.Comm., 167: 1341-1347 (1990). Rella et al., Ital. J. Biochem., 39: 83-99(1990). Forterre et al., Can. J. Microbiol., 35: 228-233 (1989). Rossiet al., System. Appl. Microbiol., 7: 337-341 (1986). Klimczak et al.,Nucleic Acids Res., 13: 5269-5282 (1985). Elie et al., Biochim. Biophys.Acta 951: 261-267 (1988). Bacillus caldotenax J. Biochem., 113: 401-410(1993). Bacillus stearothermophilus Sellmann et al., J. Bacteriol., 174:4350-4355 (1992). Stenesh and McGowan, Biochim. Biophys. Acta 475: 32-44(1977). Stenesh and Roe, Biochim. Biophys. Acta 272: 156-166 (1972).Kaboev et al., J. Bacteriol., 145: 21-26 (1981). MethanobacteriumKlimczak et al., Biochem., 25: 4850-4855 (1986). thermoautotropicumThermococcus litoralis Kong et al., J. Biol. Chem. 268: 1965 (1993) U.S.Pat. No. 5,210,036* U.S. Pat. No. 5,322,785* Anaerocellum thermophilusAnkenbauer et al., WO 98/14588* Pyrococcus sp. KOD1 U.S. Pat. No.6,008,025* Pyrococcus furiosus Lundberg et al., Gene 108: 1 (1991) PCTPub. WO 92/09689* U.S. Pat. No. 5,948,663* U.S. Pat. No. 5,866,395**Herein incorporated by reference.

These enzymes can be used at higher temperatures than retroviral reversetranscriptases so that much of the secondary structure of RNA moleculesis removed. However, the reverse transcriptase activity of many of thesepolymerases is only observed in the presence of manganese ions. Reversetranscription reactions conducted in the presence of manganese are oftensuboptimal because the presence of manganese ions lowers the fidelity ofthe polymerase and can cause damage to polynucleotides. To date, only asmall subset of thermostable DNA polymerases and mixtures have beenshown to have reverse transcriptase activity in the presence ofmagnesium ions: A. thermophilum DNA polymerase (e.g., WO 98/14588,incorporated herein by reference); B. caldotenax DNA polymerase (e.g.,U.S. Pat. No. 5,436,149, incorporated herein by reference); and thepolymerase mixture marketed as C. THERM (Boehringer Mannheim).

In the present invention, thermostable stable polymerases were screenedfor their ability to reverse transcribe a RNA template in the presenceof magnesium ions. While more than ten polymerases were screened, onlythree (i.e., T. vulgaris, B. stearothermophilus, and A. thermophilum DNApolymerases) demonstrated reverse transcriptase activity in the presenceof magnesium ions. Under the conditions utilized herein, Tvu and Bst DNApolymerases demonstrated greater than 50-fold higher reversetranscriptase activity in the presence of Mg²⁺ than native Taq DNApolymerase, sequencing-grade Taq DNA polymerase, Tth DNA polymerase, andTne DNA polymerase.

Reverse transcription of a RNA template into cDNA is an integral part ofmany techniques used in molecular biology. Accordingly, the reversetranscription procedures, mixtures, and kits provided in the presentinvention find a wide variety of uses. For example, it is contemplatedthat the reverse transcription procedures and compositions of thepresent invention are utilized to produce cDNA inserts for cloning intocDNA library vectors (e.g., lambda gt10[Huynh et al., In DNA CloningTechniques: A Practical Approach, D. Glover, ed., IRL Press, Oxford, 49,1985], lambda gt11 [Young and Davis, Proc. Nat'l. Acad. Sci., 80:1194,1983), pBR322 [Watson, Gene 70:399-403, 1988], pUC19 [Yarnisch-Perron etal., Gene 33:103-119, 1985], and M13 [Messing et al., Nucl. Acids. Res.9:309-321, 1981]). The present invention also finds use foridentification of target RNAs in a sample via RT-PCR (e.g., U.S. Pat.No. 5,322,770, incorporated herein by reference). Additionally, thepresent invention finds use in providing cDNA templates for techniquessuch as differential display PCR (e.g., Liang and Pardee, Science257(5072):967-71 (1992).

The following description of the invention is divided into: I.Thermostable DNA Polymerase Compositions; II. Use of Thermostable DNAPolymerases for Reverse Transcription; III. Use of Thermostable DNApolymerases for RT-PCR; and IV. Kits for Reverse Transcription.

I. Thermostable DNA Polymerase Compositions

In some embodiments of the present invention, compositions forperforming reverse transcription and RT-PCR are provided. Those skilledin the art will recognize that the concentrations or amounts of many ofthese components is varied for particular circumstances. For example,the optimum primer and divalent salt concentrations are known to varyfor different primers or primer pairs. Therefore, the concentrations andamounts of composition components listed below are meant to serve asguide to those skilled in the art, and are not intended to limit thescope of the invention.

A. DNA Polymerases

In some embodiments, the compositions include a thermostable DNApolymerase (e.g., Tvu DNA polymerase or Bst DNA polymerase (New EnglandBiolabs, Beverley, Mass.). In some embodiments, the Tvu DNA polymeraseis encoded by an amino acid sequence selected from SEQ ID NOs: 2, 4, and6. In other embodiments, the Bst DNA polymerase is encoded by SEQ ID NO:19. In other embodiments, the Bst DNA polymerase has reduced 5′ or 3′exonuclease activity (SEQ ID NOs. 20 and 21; e.g., U.S. Pat. Nos.5,747,298, 5,834,253, 5,874,282, and 5,830,714, incorporated herein byreference).

The present invention provides wild-type and mutant forms of Tvu DNApolymerases. In preferred embodiments, the mutant forms aresubstantially free of 5′ to 3′ exonuclease activity. Without beinglimited to any particular mutant, representative examples of mutant TvuDNA polymerases are provided herein. M285 (SEQ ID NO: 4) begins at themethionine codon located at residue 285 of the wild type Tvu DNApolymerase and ends at the wild type termination codon. M285 is encodedby the nucleic acid sequence of SEQ ID NO: 3. T289M (SEQ ID NO: 6)begins at residue 289 of the wild type Tvu DNA polymerase which wasmutated from a threonine to a methionine and ends at the wild typetermination codon. T289M is encoded by the nucleic acid sequence of SEQID NO: 5. The modified Tvu polymerases of the present invention areadvantageous in situations where the polymerization (i.e., synthetic)activity of the enzyme is desired but the presence of 5′ to 3′exonuclease activity is not.

The present invention is not intended to be limited by the nature of thealteration (e.g., deletion, insertion, substitution) necessary to renderthe Tvu polymerase deficient in 5′ to 3′ exonuclease activity. Indeed,the present invention contemplates a variety of methods, including butnot limited to proteolysis and genetic manipulation.

The present invention provides nucleic acids encoding Tvu DNA polymeraseI (SEQ ID NO: 1). Other embodiments of the present invention providepolynucleotide sequences that are capable of hybridizing to SEQ ID NO: 1under conditions of high stringency. In some embodiments, thehybridizing polynucleotide sequence encodes a protein that retains atleast one biological activity of the naturally occurring Tvu DNApolymerase. In preferred embodiments, hybridization conditions are basedon the melting temperature (T_(m)) of the nucleic acid binding complexand confer a defined “stringency” as explained above (e.g., Wahl, etal., Methods Enzymol., 152:399-407, 1987, incorporated herein byreference).

In other embodiments of the present invention, variants of Tvu DNApolymerase are provided (e.g. SEQ ID NOs: 3 and 5). In preferredembodiments, variants result from mutation, (i.e., a change in thenucleic acid sequence) and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one, or many variant forms. Common mutationalchanges that give rise to variants are generally ascribed to deletions,additions or substitutions of nucleic acids. Each of these types ofchanges may occur alone, or in combination with the others, and at therate of one or more times in a given sequence.

In still other embodiments, the nucleotide sequences of the presentinvention may be engineered in order to alter a Tvu DNA polymerasecoding sequence including, but not limited to, alterations that modifythe cloning, processing, localization, secretion, and/or expression ofthe gene product. For example, mutations may be introduced usingtechniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, alter glycosylationpatterns, or change codon preference, etc.).

In other embodiments, the present invention provides Tvu DNA polymerasepolypeptide (e.g., SEQ ID NO: 2). Other embodiments of the presentinvention provide fragments, fusion proteins or functional equivalentsof Tvu DNA polymerase (e.g., SEQ ID NOs: 4, 6). In still otherembodiments of the present invention, nucleic acid sequencescorresponding to Tvu DNA polymerase may be used to generate recombinantDNA molecules that direct the expression of Tvu DNA polymerase andvariants in appropriate host cells. In some embodiments of the presentinvention, the polypeptide may be a naturally purified product, while inother embodiments it may be a product of chemical synthetic procedures,and in still other embodiments it may be produced by recombinanttechniques using a prokaryotic or eukaryotic host cell (e.g., bybacterial cells in culture). In other embodiments, the polypeptides ofthe invention may also include an initial methionine amino acid residue.

In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than SEQ ID NO: 1encoding substantially the same or a functionally equivalent amino acidsequence, may be used to clone and express Tvu DNA polymerase. Ingeneral, such polynucleotide sequences hybridize to SEQ ID NO: 1 underconditions of medium stringency as described above. As will beunderstood by those of skill in the art, it may be advantageous toproduce Tvu DNA polymerase-encoding nucleotide sequences possessingnon-naturally occurring codons. Therefore, in some preferredembodiments, codons preferred by a particular prokaryotic or eukaryotichost (Murray et al., Nuc Acids Res 17, 1989) are selected, for example,to increase the rate of Tvu DNA polymerase expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life than transcripts produced from naturally occurringsequence.

1. Vectors for Production of Tvu DNA Polymerase

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NOs: 1, 3, 5, 18 and 20). In someembodiments of the present invention, the constructs comprise a vectorinto which a sequence of the invention has been inserted, in a forwardor reverse orientation. In still other embodiments, the heterologousstructural sequence (e.g., SEQ ID NOs: 1, 3, 5, 18, and 20) is assembledin appropriate phase with translation initiation and terminationsequences. In preferred embodiments of the present invention, theappropriate DNA sequence is inserted into the vector using any of avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart.

Large numbers of suitable vectors that are replicable and viable in thehost are known to those of skill in the art, and are commerciallyavailable. Any plasmid or vector may be used as long as it is replicableand viable in the host. In some preferred embodiments of the presentinvention, bacterial expression vectors comprise an origin ofreplication, a suitable promoter and optionally an enhancer, and alsoany necessary ribosome binding sites, polyadenylation sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences.

In certain embodiments of the present invention, the Tvu DNA sequence inthe expression vector is operatively linked to an appropriate expressioncontrol sequence(s) (e.g., a constitutive or inducible promoter) todirect mRNA synthesis. Promoters useful in the present inventioninclude, but are not limited to, a retroviral LTR, SV40 promoter, CMVpromoter, RSV promoter, E. coli lac or trp promoters, phage lambda P_(L)and P_(R) promoters, T3, SP6 and T7 promoters. In other embodiments ofthe present invention, recombinant expression vectors include origins ofreplication and selectable markers, (e.g., tetracycline or ampicillinresistance in E. coli, or neomycin phosphotransferase gene for selectionin eukaryotic cells).

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation, as well as a transcriptionterminator. In still other embodiments of the present invention, thevector may also include appropriate sequences for enhancing expression.

2. Host Cells and Systems for Production of Tvu DNA Polymerase

The present invention contemplates that the nucleic acid construct ofthe present invention be capable of expression in a suitable host. Inparticular, it is preferable that the expression system chosen utilizesa controlled promoter such that expression of the Tvu polymerase isprevented until expression is induced. In this manner, potentialproblems of toxicity of the expressed polymerases to the host cells (andparticularly to bacterial host cells) are avoided. Those in the art knowmethods for attaching various promoters and 3′ sequences to a genesequence in order to achieve efficient and tightly controlledexpression. The examples below disclose a number of suitable vectors andvector constructs. Of course, there are other suitable promoter/vectorcombinations. The choice of a particular vector is also a function ofthe type of host cell to be employed (i.e., prokaryotic or eukaryotic).

In some embodiments of the present invention, the host cell can be aprokaryotic cell (e.g., a bacterial cell). Specific examples of hostcells include, but are not limited to, E. coli, S. typhimurium, B.subtilis, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by any suitable method known in the art (e.g., calciumphosphate transfection, DEAE-Dextran mediated transfection, orelectroporation (e.g., Davis et al., Basic Methods in Molecular Biology,1986, for a review). Alternatively, in some embodiments of the presentinvention, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction), andthe host cells are cultured for an additional period. In otherembodiments of the present invention, the host cells are harvested(e.g., by centrifugation), disrupted by physical or chemical means, andthe resulting crude extract retained for further purification. In stillother embodiments of the present invention, microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption, or useof cell lysing agents.

It is not necessary that a host organism be used for the expression ofthe nucleic acid constructs of the invention. For example, expression ofthe protein encoded by a nucleic acid construct may be achieved throughthe use of a cell-free in vitro transcription/translation system. Anexample of such a cell-free system is the commercially available TnT™Coupled Reticulocyte Lysate System (Promega; this cell-free system isdescribed in U.S. Pat. No. 5,324,637, herein incorporated by reference).

3. Purification of Tvu DNA Polymerase

The present invention also provides methods for recovering and purifyingTvu DNA polymerase from native and recombinant cell cultures including,but not limited to, ammonium sulfate precipitation, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. In other embodiments of thepresent invention, protein refolding steps can be used as necessary, incompleting configuration of the mature protein. In still otherembodiments of the present invention, high performance liquidchromatography (HPLC) can be employed as one or more purification steps.In some embodiments, purification is performed as described in Example1.

In other embodiments of the present invention, the nucleic acidconstruct containing DNA encoding the wild-type or a variant Tvupolymerase further comprises the addition of exogenous sequences (i.e.,sequences not encoded by the Tvu polymerase coding region) to either the5′ or 3′ end of the Tvu polymerase coding region to allow for ease inpurification of the resulting polymerase protein (the resulting proteincontaining such an affinity tag is termed a “fusion protein”). Severalcommercially available expression vectors are available for attachingaffinity tags (e.g., an exogenous sequence) to either the amino orcarboxy-termini of a coding region. In general these affinity tags areshort stretches of amino acids that do not alter the characteristics ofthe protein to be expressed (i.e., no change to enzymatic activitiesresults).

For example, the pET expression system (Novagen) utilizes a vectorcontaining the T7 promoter operably linked to a fusion protein with ashort stretch of histidine residues at either end of the protein and ahost cell that can be induced to express the T7 DNA polymerase (i.e., aDE3 host strain). The production of fusion proteins containing ahistidine tract is not limited to the use of a particular expressionvector and host strain. Several commercially available expressionvectors and host strains can be used to express protein sequences as afusion protein containing a histidine tract (e.g., the pQE series[pQE-8, 12, 16, 17, 18, 30, 31, 32, 40, 41, 42, 50, 51, 52, 60 and 70]of expression vectors (Qiagen) used with host strains M15[pREP4][Qiagen] and SG13009[pREP4] [Qiagen]) can be used to express fusionproteins containing six histidine residues at the amino-terminus of thefusion protein). Additional expression systems which utilize otheraffinity tags are known to the art.

Once a suitable nucleic acid construct has been made, the Tvu DNApolymerase may be produced from the construct. The examples below andstandard molecular biological teachings known in the art enable one tomanipulate the construct by a variety of suitable methods. Once thedesired Tvu polymerase has been expressed, the polymerase may be testedfor DNA synthesis as described below.

4. Deletion Mutants of Tvu DNA Polymerase

The present invention further provides fragments of Tvu DNA polymerase(i.e., deletion mutants; e.g., SEQ ID NOs 4 and 6). In some embodimentsof the present invention, when expression of a portion of Tvu DNApolymerase is desired, it may be necessary to add a start codon (ATG) tothe oligonucleotide fragment containing the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al., J. Bacteriol., 169:751-757, 1987) and S.typhimurium, and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al., Proc. Nat'l. Acad. Sci.,84:2718-1722, 1990). Therefore, removal of an N-terminal methionine, ifdesired, can be achieved either in vivo by expressing such recombinantpolypeptides in a host producing MAP (e.g., E. coli or CM89 or S.cerevisiae), or in vitro by use of purified MAP.

In other embodiments of the present invention, Tvu DNA polymeraseshaving a reduced level of 5′ to 3′ exonuclease compared to wild-typewere produced by subcloning portions of Tvu DNA polymerase lacking the5′ to 3′ exonuclease-encoding domain (Examples 11-12). In otherembodiments, proteolysis is used to remove portion of Tvu polymeraseresponsible for 5′ to 3′ exonuclease activity. Following proteolyticdigestion, the resulting fragments are separated by standardchomatographic techniques and assayed for the ability to synthesize DNAand to act as a 5′ to 3′ exonuclease.

5. Variants of Tvu DNA Polymerase

Still other embodiments of the present invention provide other mutant orvariant forms of Tvu DNA polymerase. It is possible to modify thestructure of a peptide having an activity (e.g., DNA synthesis activity)of Tvu DNA polymerase for such purposes as enhancing stability (e.g., invitro shelf life, and/or resistance to proteolytic degradation in vivo)or reducing 5′ to 3′ exonuclease activity. Such modified peptides areconsidered functional equivalents of peptides having an activity of TvuDNA polymerase as defined herein. A modified peptide can be produced inwhich the nucleotide sequence encoding the polypeptide has been altered,such as by substitution, deletion, or addition. In some preferredembodiments of the present invention, the alteration decreases the 5′ to3′ exonuclease activity to a level low enough to provide an improvedenzyme for a variety of applications such as PCR and chain terminationsequencing (including thermal cycle sequencing) as discussed in theExamples below. In particularly preferred embodiments, thesemodifications do not significantly reduce the DNA synthesis activity ofthe modified enzyme. In other words, construct “X” can be evaluatedaccording to the protocol described below in order to determine whetherit is a member of the genus of modified Tvu polymerases of the presentinvention as defined functionally, rather than structurally.

Moreover, as described above, variant forms of Tvu DNA polymerase arealso contemplated as being equivalent to those peptides and DNAmolecules that are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of Tvu DNApolymerase containing conservative replacements. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare can be divided into four families: (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes, e.g.,replacement of a glycine with a tryptophan. Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs well known in the art, for example LASERGENE software(DNASTAR Inc., Madison, Wis.).

When a Tvu DNA polymerase enzyme of the present invention has an aminoacid residue sequence that is not identical to that of SEQ ID NOs: 2, 4or 6 because one or more conservative substitutions has been made, it ispreferred that no more than 20 percent, and more preferably no more than10 percent, and most preferably no more than 5 percent of the amino acidresidues are substituted as compared to SEQ ID NOs: 2, 4 or 6.

A contemplated Tvu DNA polymerase can also have a length shorter thanthat of SEQ ID NO: 2 and maintain DNA synthesis activity. For example,the first 284 amino acids at the amino terminus can be deleted as in anenzymes of SEQ ID NO: 4 and 6. Such variants exhibit DNA synthesisactivity as discussed elsewhere herein, including DNA synthesis activityat temperatures higher than about 50° C.

This invention further contemplates a method for generating sets ofcombinatorial mutants of the present Tvu DNA polymerase, as well asdeletion mutants, and is especially useful for identifying potentialvariant sequences (i.e., homologs) with unique DNA synthetic activity.The purpose of screening such combinatorial libraries is to generate,for example, novel Tvu DNA polymerase homologs that possess novelactivities.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of Tvu DNApolymerase homologs or other related proteins are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, DNA polymerase homologs from one or more species,or Tvu DNA polymerase homologs from the same species but which differdue to mutation. Amino acids appearing at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences.

In a preferred embodiment of the present invention, the combinatorialTvu DNA polymerase library is produced by way of a degenerate library ofgenes encoding a library of polypeptides including at least a portion ofpotential Tvu DNA polymerase-protein sequences. For example, a mixtureof synthetic oligonucleotides are enzymatically ligated into genesequences such that the degenerate set of potential Tvu DNA polymerasesequences are expressible as individual polypeptides, or alternatively,as a set of larger fusion proteins (e.g., for phage display) containingthe set of Tvu DNA polymerase sequences therein.

There are many ways in which the library of potential Tvu DNA polymerasehomologs can be generated from a degenerate oligonucleotide sequence. Insome embodiments, chemical synthesis of a degenerate gene sequence iscarried out in an automatic DNA synthesizer, and the synthetic genes areligated into an appropriate gene for expression. The purpose of adegenerate set of genes is to provide, in one mixture, all of thesequences encoding the desired set of potential Tvu DNA polymerasesequences. The synthesis of degenerate oligonucleotides is well known inthe art (e.g., Narang, S. A, Tetrahedron 39:3 9, 1983; Itakura et al.,Recombinant DNA, Proc 3rd Cleveland Sympos. Macromol., Walton, ed.,Elsevier, Amsterdam, pp 273-289, 1981; Itakura et al., Annu. Rev.Biochem. 53:323, 1984; Itakura et al., Science 198:1056, 1984; and Ikeet al., Nucleic Acid Res. 11:477 1983). Such techniques have beenemployed in the directed evolution of other proteins (e.g., Scott etal., Science 249:386-390, 1980; Roberts et al., Proc. Nat'l. Acad. Sci.,89:2429-2433, 1992; Devlin et al., Science 249: 404-406, 1990; Cwirla etal., Proc. Nat'l. Acad. Sci., 87: 6378-6382, 1990; as well as U.S. Pat.Nos. 5,223,409, 5,198,346, and 5,096,815, each of which is incorporatedherein by reference).

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries generated by point mutations, andfor screening cDNA libraries for gene products having a particularproperty of interest. Such techniques are generally adaptable for rapidscreening of gene libraries generated by the combinatorial mutagenesisof Tvu DNA polymerase homologs. The most widely used techniques forscreening large gene libraries typically comprise cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions such that detection of a desiredactivity facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. The illustrative assays describedbelow are amenable to high through-put analysis as necessary to screenlarge numbers of degenerate sequences created by combinatorialmutagenesis techniques.

In some embodiments of the present invention, the gene library isexpressed as a fusion protein on the surface of a viral particle. Forexample, foreign peptide sequences can be expressed on the surface ofinfectious phage in the filamentous phage system, thereby conferring twosignificant benefits. First, since these phage can be applied toaffinity matrices at very high concentrations, a large number of phagecan be screened at one time. Second, since each infectious phagedisplays the combinatorial gene product on its surface, if a particularphage is recovered from an affinity matrix in low yield, the phage canbe amplified by another round of viral replication. The group of almostidentical E. coli filamentous phages M13, fd, and fl are most often usedin phage display libraries, as either of the phage gIII or gVIII coatproteins can be used to generate fusion proteins without disrupting theultimate packaging of the viral particle (e.g., WO 90/02909; WO92/09690; Marks et al., J. Biol. Chem., 267:16007-16010, 1992; Griffthset al., EMBO J., 12:725-734, 1993; Clackson et al., Nature, 352:624-628,1991; and Barbas et al., Proc. Nat'l. Acad. Sci., 89:4457-4461, 1992).

In another embodiment of the present invention, the recombinant phageantibody system (e.g., RPAS, Pharmacia Catalog number 27-9400-01) ismodified for use in expressing and screening Tvu polymerasecombinatorial libraries. The pCANTAB 5 phagemid of the RPAS kit containsthe gene encoding the phage gIII coat protein. In some embodiments ofthe present invention, the Tvu polymerase combinatorial gene library iscloned into the phagemid adjacent to the gIII signal sequence such thatit will be expressed as a gIII fusion protein. In other embodiments ofthe present invention, the phagemid is used to transform competent E.coli TG1 cells after ligation. In still other embodiments of the presentinvention, transformed cells are subsequently infected with M13KO7helper phage to rescue the phagemid and its candidate Tvu polymerasegene insert. The resulting recombinant phage contain phagemid DNAencoding a specific candidate Tvu polymerase-protein and display one ormore copies of the corresponding fusion coat protein. In someembodiments of the present invention, the phage-displayed candidateproteins that are capable of, for example, binding nucleotides ornucleic acids, are selected or enriched by panning. The bound phage isthen isolated, and if the recombinant phage express at least one copy ofthe wild type gIII coat protein, they will retain their ability toinfect E. coli. Thus, successive rounds of reinfection of E. coli andpanning greatly enriches for Tvu polymerase homologs, which are thenscreened for further biological activities.

In light of the present disclosure, other forms of mutagenesis generallyapplicable will be apparent to those skilled in the art in addition tothe aforementioned rational mutagenesis based on conserved versusnon-conserved residues. For example, Tvu DNA polymerase homologs can begenerated and screened using, for example, alanine scanning mutagenesisand the like (Ruf et al., Biochem., 33:1565-1572, 1994; Wang et al., J.Biol. Chem., 269:3095-3099, 1994; Balint et al. Gene 137:109-118, 1993;Grodberg et al., Eur. J. Biochem., 218:597-601, 1993; Nagashima et al.,J. Biol. Chem., 268:2888-2892, 1993; Lowman et al., Biochem.,30:10832-10838, 1991; and Cunningham et al., Science, 244:1081-1085,1989); linker scanning mutagenesis (Gustin et al., Virol., 193:653-660,1993; Brown et al., Mol. Cell. Biol., 12:2644-2652, 1992; McKnight etal., Science, 232:316); or saturation mutagenesis (Meyers et al.,Science, 232:613, 1986).

In some embodiments, the wild-type Tvu polymerase is cloned by isolatinggenomic DNA using molecular biological methods. The isolated genomic DNAis then cleaved into fragments (e.g., about 3 kb or larger) usingrestriction enzymes and the fragments are inserted into a suitablecloning vector such as a plasmid or bacteriophage vector. The vectorscontaining fragments of T. vulgaris genomic DNA are then transformedinto a suitable E. coli host. Clones containing DNA encoding the Tvupolymerase may be isolated using functional assays (e.g., presence ofthermostable polymerase in lysates of transformed cells) or byhybridization using a probe derived from a region of conservation amongDNA polymerases derived from thermostable organisms. Alternatively, theT. vulgaris genomic DNA may be used as the target in PCR with primersselected from regions of high sequence conservation among the genesencoding thermostable DNA polymerases. Although such a PCR may notamplify the entire coding region of the Tvu polymerase I gene, thefull-length Tvu gene can be isolated by using the amplified fragment asa probe to screen a genomic library containing T. vulgaris DNA.

Once the full-length Tvu polymerase gene is obtained, the regionencoding the 5′ to 3′ exonuclease may be altered by a variety of meansto reduce or eliminate these activities. Suitable deletion andsite-directed mutagenesis procedures are known in the art.

In some embodiments of the present invention, deletion of amino acidsfrom the protein is accomplished either by deletion in the encodinggenetic material, or by introduction of a translational stop codon bymutation or frame shift. In other embodiments, proteolytic treatment ofthe protein molecule is performed to remove portions of the protein. Instill further embodiments, deletion mutants are constructed byrestriction digesting the wild-type sequence and introducing a new startsite by annealing an appropriately designed oligomer to the digestedfragment encoding the desired activity (e.g., Example 11).

6. Chemical Synthesis of Tvu DNA Polymerase

In an alternate embodiment of the invention, the coding sequence of TvuDNA polymerase is synthesized, whole or in part, using chemical methodswell known in the art (e.g., Caruthers et al., Nuc. Acids Res. Symp.Ser., 7:215-233, 1980; Crea and Horn, Nuc. Acids Res., 9:2331, 1980;Matteucci and Caruthers, Tetrahedron Lett., 21:719, 1980; and Chow andKempe, Nuc. Acids Res., 9:2807-2817, 1981). In other embodiments of thepresent invention, the protein itself is produced using chemical methodsto synthesize either a full-length Tvu DNA polymerase amino acidsequence or a portion thereof. For example, peptides can be synthesizedby solid phase techniques, cleaved from the resin, and purified bypreparative high performance liquid chromatography (e.g., Creighton,Proteins Structures and Molecular Principles, W H Freeman and Co, NewYork N.Y. 1983, for a review). In other embodiments of the presentinvention, the composition of the synthetic peptides is confirmed byamino acid analysis or sequencing (e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al., Science 269:202-204, 1995) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequence of Tvu DNApolymerase, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with other sequences to produce avariant polypeptide.

B. Other Components

The present invention also provides compositions for performing variousreactions (e.g., reverse transcription, polymerase chain reaction,sequencing, first strand cDNA synthesis, and second strand cDNAsynthesis) with Tvu and Bst polymerases. In other embodiments, thecomposition further comprises an additional thermostable polymerase(e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, andthe like). In some embodiments, the compositions include a purified RNAtemplate (e.g., mRNA, rRNA, and mixtures thereof). In other embodiments,the compositions include a buffering agent (e.g., Tris-HCl) at aconcentration of about 5 mM to 100 mM, preferably about 10 mM. Infurther embodiments, the pH of the buffer is from about 6.0 to 10.0,preferably about 7.0 to 9.0. In still further embodiments, thecomposition includes a monovalent salt (e.g., NaCl or KCl at aconcentration of about 10 mM to 100 mM, preferably about 50 mM). Instill further embodiments, the composition includes a divalent salt. Insome embodiments, the divalent salt is MgCl₂ at a concentration of about0.5 mM to 25 mM, preferably about 1.5 mM to about 10 mM. In otherembodiments, the divalent salt is MnCl₂ at a concentration of about 0.1mM to about 10 mM, preferably about 0.6 mM. In still other embodiments,the composition is substantially manganese ion free. In furtherembodiments, the composition includes deoxynucleotide phosphates (dNTPs)at a concentration of about 0.5 to 5 mM each, preferably about 0.2 mMeach. In still further embodiments, the composition includes one or moreprimers, preferably at a concentration of about 0.1 to 5 μM. In otherembodiments, the compositions includes additives that serve to increasethe stability of the components of the reaction (e.g., a cationic ornon-ionic surfactant) or to increase the efficiency of amplification(e.g., formamide or betaine).

II. Use of Thermostable DNA Polymerases for Reverse Transcription

The present invention contemplates the use of Tvu and Bst DNA polymerasefor reverse transcription reactions. Accordingly, in some embodiments ofthe present invention, thermostable DNA polymerases having reversetranscriptase activity are provided. In some embodiments, thethermostable DNA polymerase is selected from Tvu DNA polymerase and BstDNA polymerase. In further embodiments, the reverse transcriptaseactivity is exhibited in the presence of magnesium or manganese ions. Inother embodiments, the polymerase exhibits reverse transcriptaseactivity in the presence of magnesium ions and the substantial absenceof manganese ions.

In some embodiments of the present invention, where Tvu polymerase isutilized to reverse transcribe RNA, the reverse transcription reactionis conducted at about 50° C. to 80° C., preferably about 60° C. to 75°C. In embodiments where Bst reverse transcriptase is utilized forreverse transcription, the reaction is conducted at 50° C. to 75° C.,preferably at about 60° C. to 70° C.

In still further embodiments, reverse transcription of an RNA moleculeby Tvu or Bst DNA polymerase results in the production of a cDNAmolecule that is substantially complementary to the RNA molecule. Inother embodiments, the Tvu or Bst DNA polymerase then catalyzes thesynthesis of a second strand DNA complementary to the cDNA molecule toform a double stranded DNA molecule. In still further embodiments of thepresent invention, the Tvu polymerase catalyzes the amplification of thedouble stranded DNA molecule in a PCR as described above. In someembodiments, PCR is conducted in the same reaction mix as the reversetranscriptase reaction (i.e., a single pot reaction is performed). WhileTvu DNA polymerase and Bst DNA polymerase are suitable for use in somesingle pot reactions, the data presented in the Examples indicate thatthe inclusion of an additional DNA polymerase is preferable for mostamplification procedures due to the lower temperature optimums for TvuDNA polymerase and Bst DNA polymerase than for other polymerases such asTaq, Pfu and the like. Therefore, in some embodiments, the reactionmixture further comprises an additional thermostable polymerase (e.g.,Taq DNA polymerase, Tne DNA polymerase, Pfu DNA polymerase, and thelike). In other embodiments, PCR is performed in a separate reaction mixon an aliquot removed from the reverse transcription reaction (i.e., atwo pot reaction is performed).

III. Use of Thermostable DNA Polymerases for RT-PCR

The DNA polymerases of the present invention are useful for RT-PCRbecause the reverse transcription reaction may be conducted in amagnesium-containing buffer that is compatible with efficientamplification. The present invention contemplates single-reaction RT-PCRwherein reverse transcription and amplification are performed in asingle, continuous procedure. The RT-PCR reactions of the presentinvention serve as the basis for many techniques, including, but notlimited to diagnostic techniques for analyzing mRNA expression,synthesis of cDNA libraries, rapid amplification of cDNA ends (i.e.,RACE) and other amplification-based techniques known in the art. Anytype of RNA may be reverse transcribed and amplified by the methods andreagents of the present invention, including, but not limited to RNA,rRNA, and mRNA. The RNA may be from any source, including, but notlimited to, bacteria, viruses, fungi, protozoa, yeast, plants, animals,blood, tissues, and in vitro synthesized nucleic acids.

The wild-type and modified Tvu and Bst DNA polymerases of the presentinvention provide suitable enzymes for use in the PCR. The PCR processis described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosuresof which are incorporated herein by reference. In some embodiments, atleast one specific nucleic acid sequence contained in a nucleic acid ormixture of nucleic acids is amplified to produce double stranded DNA.Primers, template, nucleoside triphosphates, the appropriate buffer andreaction conditions, and polymerase are used in the PCR process, whichinvolves denaturation of target DNA, hybridization of primers andsynthesis of complementary strands. The extension product of each primerbecomes a template for the production of the desired nucleic acidsequence. If the polymerase employed in the PCR is a thermostableenzyme, then polymerase need not be added after each denaturation stepbecause heat will not destroy the polymerase activity. Use of suchenzymes as Tvu or Bst DNA DNA polymerase allows repetitiveheating/cooling cycles without the requirement of fresh enzyme at eachcooling step. This represents a major advantage over the use ofmesophilic enzymes (e.g., Klenow), as fresh enzyme must be added to eachindividual reaction tube at every cooling step. The use of Taq in PCR isdisclosed in U.S. Pat. No. 4,965,188, EP Publ. No. 258,017, and PCTPubl. No. 89/06691, each of which is herein incorporated by reference.

RT-PCR may be divided into two main steps, reverse transcription of RNAto form a cDNA and amplification of the cDNA. In most prior art RT-PCRmethods, these two main steps are separate, with one enzyme being usedfor reverse transcription, and a different DNA polymerase being used foramplification. In most cases, even when the same enzyme is used forreverse transcription and amplification, some purification of thereverse transcription step product was necessary because of theincompatibility of the buffers used for reverse transcription andamplification.

In some embodiments of the present invention, the reverse transcriptionand amplification steps of RT-PCR are conducted in the same buffer. Infurther embodiments, reverse transcription of an RNA into a cDNA, secondstrand synthesis of a copy of the cDNA, and amplification of the cDNAare conducted in a continuous process in the same reaction mix. In somepreferred embodiments, the single pot reaction mixture further comprisesan additional thermostable polymerase (e.g., Taq DNA polymerase, Tne DNApolymerase, Pfu DNA polymerase, and the like). In some embodiments, thebuffer comprises magnesium and/or manganese ions. In other embodiments,the buffer comprises magnesium ions. In other embodiments, the buffer issubstantially free of manganese ions. In still other embodiments, thereverse transcription step is performed at an elevated temperature asdescribed above. In some embodiments of the present invention, primersfor reverse transcription also serve as primers for amplification. Inother embodiments, the primer or primers used for reverse transcriptionare different than the primers used for amplification. In someembodiments, more than one RNA in a mixture of RNAs may be amplified ordetected by RT-PCR. In other embodiments, multiple RNAs in a mixture ofRNAs may be amplified in a multiplex procedure (e.g., U.S. Pat. No.5,843,660, incorporated herein by reference). In still furtherembodiments of the present invention, the reverse transcription reactionis performed with Tvu or Bst DNA polymerase, while the amplificationstep is performed with another thermostable DNA polymerase (e.g., TthDNA polymerase, Taq DNA polymerase, or Tne DNA polymerase). In stillother embodiments, the reverse transcription reaction is performed withone enzyme (e.g., MMLV or AMV), while the amplification reaction isperformed with Tvu or Bst DNA polymerase.

IV. Kits for Reverse Transcription.

In other embodiments of the present invention, kits are provided forperforming reverse transcription. It is contemplated that the kits ofthe present invention find use for methods including, but not limitedto, reverse transcribing template RNA for the construction of cDNAlibraries, for the reverse transcription of RNA for differential displayPCR, and RT-PCR identification of target RNA in a sample suspected ofcontaining the target RNA. In some embodiments, the reversetranscription kit comprises the essential reagents required for themethod of reverse transcription. For example, in some embodiments, thekit includes a vessel containing a polymerase selected from T. vulgarisand B. stearothermophilus polymerase. In some embodiments, the kitfurther comprises a container containing an additional thermostablepolymerase (e.g., Taq DNA polymerase, Tne DNA polymerase, Pfu DNApolymerase, and the like). In some embodiments, the concentration ofpolymerase ranges from about 0.1 to 100 u/μl; in other embodiments, theconcentration is about 5 u/μl. In some embodiments, kits for reversetranscription also include a vessel containing a reaction buffer.Preferably, these reagents are free of contaminating RNase activity. Inother embodiments of the present invention, reaction buffers comprise abuffering reagent in a concentration of about 5 to 15 mM (preferablyabout 10 mM Tris-HCl at a pH of about 7.5 to 9.0 at 25° C.), amonovalent salt in a concentration of about 20 to 100 mM (preferablyabout 50 mM NaCl or KCl), a divalent cation in a concentration of about1.0 to 10.0 mM (preferably MgCl₂), dNTPs in a concentration of about0.05 to 1.0 mM each (preferably about 0.2 mM each), and a surfactant ina concentration of about 0.001 to 1.0% by volume (preferably about 0.01%to 0.1%). In some embodiments, a purified RNA standard set is providedin order to allow quality control and for comparison to experimentalsamples. In some embodiments, the kit is packaged in a single enclosureincluding instructions for performing the assay methods (e.g., reversetranscription or RT-PCR). In some embodiments, the reagents are providedin containers and are of a strength suitable for direct use or use afterdilution.

EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: ° C.(degrees Centigrade); g (gravitational field); vol (volume); w/v (weightto volume); v/v (volume to volume); BSA (bovine serum albumin); CTAB(cetyltrimethylammonium bromide); fmol (femtomole); HPLC (high pressureliquid chromatography); DTT (dithiothreitol); DMF (N,N dimethylformamide); DNA (deoxyribonucleic acid); p (plasmid); μl (microliters);ml (milliliters); μg (micrograms); pmoles (picomoles); mg (milligrams);MOPS (3-[N-Morpholino]propanesulfonic acid); M (molar); mM (milliMolar);μM (microMolar); nm (nanometers); kd (kilodaltons); OD (opticaldensity); EDTA (ethylene diamine tetra-acetic acid); FITC (fluoresceinisothiocyanate); SDS (sodium dodecyl sulfate); NaPO₄ (sodium phosphate);Tris(tris(hydroxymethyl)-aminomethane); PMSF(phenylmethylsulfonylfluoride); TBE (Tris-Borate-EDTA, i.e., Tris buffertitrated with boric acid rather than HCl and containing EDTA); PBS(phosphate buffered saline); PPBS (phosphate buffered saline containing1 mM PMSF); PAGE (polyacrylamide gel electrophoresis); SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis); Tween(polyoxyethylene-sorbitan); Boehringer Mannheim or BM (BoehringerMannheim, Indianapolis, Ind.); Epicentre (Epicentre Technologies,Madison, Wis.); New England Biolabs or NEB (New England Biolabs,Beverly, Mass.); Novagen (Novagen, Inc., Madison, Wis.); Pharmacia(Pharmacia Biotech Inc., Piscataway, N.J.); Perkin Elmer (Perkin Elmer,Norwalk, Conn.); Promega (Promega Corp., Madison, Wis.); Qiagen (QiagenInc., Chatsworth, Calif.); Spectra (Spectra, Houston, Tex.); Stratagene(Stratagene Cloning Systems, La Jolla, Calif.); USB (U.S. Biochemical,Cleveland, Ohio); and Tomah (Tomah Products Inc., Tomah, Wis.).

Example 1 Purification of Tvu DNA Polymerase

This example describes the purification of native T. vulgaris (Tvu) DNApolymerase. Tvu cells were obtained from the ATCC (Accession Number43649). This purified polymerase was then used in the experimentsrepresented in Examples 2 through 10. One milliliter from the frozenseed vial was thawed and inoculated into 1 liter Luria broth. The mediumwas supplemented with 10 ml of 20% glucose. The culture was grown for 15hours on a shaker at 55° C. and 250 rpm. Five hundred milliliters ofthis culture were added to 17.5 liters medium in a 20-liter fermenter.The culture was grown at 55° C. The culture growth was monitoredspectrophotometrically at 580 nm and measured based on wet weight ofcell pellets from 40 ml of broth. After 4.75 hours, the contents werechilled and harvested using a CEPA tubular bowl centrifuge. The netyield of cell paste was 69.0 g. The cell paste was stored in a freezerat −85° C., until purification of Tvu DNA polymerase was performed.

Thirty grams of cell paste were suspended in ice cold 150 ml 0.25 M NaClTEDGT buffer (50 mM Tris-HCl at pH 7.3, 1 mM EDTA, 1 mM DTT, 10%Glycerol, and 0.1% Tween 20) containing 2.5 mM PMSF, and lysed bysonication on ice. Then 11.5 ml of 5% PEI was added to the lysate toprecipitate the DNA. The following purification steps were performed at4° C. Centrifugation (15,000 rpm in a Beckman JA18 rotor for 15 minutes)was used to separate the supernatant from the precipitate. Thesupernatant was then collected, and ammonium sulfate was added to afinal saturation of 65% to precipitate the DNA polymerase.Centrifugation (15,000 rpm in a Beckman JA18 rotor for 20 minutes) wasused to separate the ammonium sulfate precipitate from the supernatant.The precipitate was collected, suspended in TEDGT buffer and dialyzedagainst TEDGT buffer to remove the ammonium sulfate.

The dialyzed solution was then loaded onto a Heparin-Agarose column (SPL1905-0004) equilibrated with TEDGT buffer. After washing the column withTEDGT buffer, elution was performed by applying a linear gradient of 0to 1 M NaCl TEDGT buffer. The fractions were collected, and assayed forDNA polymerase activity as described in Example 2. Fractions with DNApolymerase activity were pooled. The presence of endonucleases wasdetermined by incubating the equivalent of 1/64, 1/16, ⅛, ¼, ½, and 1 μlof the pooled fractions with 1 μg lambda DNA (Promega, D150) in buffer E(Promega, R005A) for one hour at 74° C. Agarose gel analysis of thedigest showed no restriction enzyme activity. The pooled fractions weredialyzed against TEDGT buffer, then loaded onto a TEDGT bufferequilibrated Cibacron Blue column (Sigma, C-1535). After washing thecolumn with TEDGT buffer, elution was performed with a linear gradientof 0 to 1 M NaCl TEDGT buffer. The eluate was collected in fractions,and each fraction was assayed for DNA polymerase activity.

Fractions that contained DNA polymerase activity were pooled, dialyzedagainst TEDGT buffer, and loaded onto a TEDGT buffer equilibratedDEAE-Sepharose column (Sigma DCL-6B-100). After washing the column withTEDGT buffer, elution was performed with a linear gradient of 0 to 1 MTEDGT buffer. The eluate was collected in fractions, and assayed for DNApolymerase activity. The fraction that showed the highest DNA polymeraseactivity was dialyzed against TEDGT buffer before it was loaded onto aTEDGT equilibrated DNA-Agarose column (Promega). After washing thecolumn with TEDGT buffer, elution was performed with a linear gradientof 0 to 1 M NaCl TEDGT buffer. The eluate was collected in fractions,and assayed for DNA polymerase activity. Endonuclease and nickaseactivities were assayed by incubating 5 μl of fractions with the highestDNA polymerase activity with 1 μg of PhiX174 DNA digested with Hae IIIrestriction enzyme (Promega, G176A) or pBR322 plasmid DNA (PromegaD151A) in buffer E (Promega R005A) for 3⅓ hours at 70° C. Fractions thatshowed highest level of DNA polymerase activity and no substantialendonuclease or nickase activity were pooled to yield a 3 ml solution.Sixty microliters 10% Tween 20 and 60 μl 10% NP40 detergents were added,and dialyzed against the storage buffer (20 mM Tris-HCl pH8.0, 100 mMKCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% NP-40, and 0.5% Tween20), diluted with the storage buffer to a concentration of 5 units (asdefined in Example 2) per microliter and stored at −20° C.

This experiment demonstrated that the Tvu DNA polymerase was purified togreater than 95% pure as indicated by the substantial absence ofnuclease contamination, and a predominant band at about 97 kD whencompared to Mark 12 size markers (Novex, LC5677) on a 4-20% Tris-Glycinegel (Novex EC6025).

Example 2 DNA Polymerization Activity Assay

Activity of native, thermostable Tvu DNA polymerase purified asdescribed in Example 1 was assayed by incorporation of radiolabeled dTTPinto nicked and gapped (i.e., activated) calf thymus DNA prepared asdescribed below. One unit of thermostable DNA polymerase is defined asthe amount of enzyme required to catalyze the incorporation of 10 nmolof dNTP into an acid-insoluble form in 30 minutes at 74° C. The reactionconditions comprised: 50 mM Tris-HCl (pH 9.0 at 25° C.), 50 mM NaCl, 10mM MgCl₂, 12 μg activated calf thymus DNA, 0.2 mM dATP, 0.2 mM dGTP, 0.2mM dCTP, 0.2 mM dTTP (Promega, U1240), and 1 μCi of ³HdTTP (Amersham,#TRK.424) per 50 μl reaction.

The reaction components were assembled at room temperature. Samplessuspected of containing polymerase activity were added (5 μl containing0.05 to 0.5 units) and the tube was incubated at 74° C. for 30 minutes.Then, 50 μl aliquots were removed at 6, 9, 12, and 15 minutes and placedin separate tubes on ice. The ³H-dTTP incorporation was determined bymeasuring TCA precipitation counts by the following procedure. To each50 μl aliquot, 500 μl 10% cold TCA solution was added and the tubes wereincubated on ice for 10 minutes before the contents of each tube werefiltered onto a separate GF/A filter (Whatman, 1820 024). The filterswere washed with 5 ml 5% cold TCA solution three times, and once withacetone. The filters were dried under a heat lamp, put into ascintillation vial, and then counted in a liquid scintillation counterin scintillation fluid (Beckman, 158735). A no-enzyme negative controlwas also performed using 50 μl DNA polymerase activity assay mix andwashed as above. The total counts of each reaction were determined using5 μl of DNA polymerase activity assay mix directly.

Activated calf thymus DNA was prepared by dissolving 1 g calf thymus DNA(#D-151, Sigma) in 400 ml TM buffer (10 mM Tris-HCl (pH 7.3), 5 mMMgCl₂). Four hundred microliters of a solution containing 40 unites ofRQ1-DNase (Promega) in TM buffer was added to the DNA solution andincubated at 37° C. for 10 minutes. The DNase digestion was stopped byheating the DNA solution at 68° C. for 30 minutes. The activated calfthymus DNA was stored at −20° C. until used. The activated calf thymusDNA was heated to 74° C. for 10 minutes and then cooled to roomtemperature before use.

Example 3 Comparison of RT Activity of Thermostable DNA Polymerases inthe Presence of Mg²⁺ or Mn²⁺ Ions

This example describes the determination of the reverse transcriptaseactivity of several different DNA polymerases in the presence of eitherMg²⁺ or Mn²⁺ ions. In these experiments, a reverse transcription (RT)reaction mix was used. The final concentration of each component in areaction was: 10 mM Tris-HCl (pH 8.3), 90 mM KCl, 0.5 mM dTTP (Promega,U123A), 0.25 mM polyriboadenylate, 0.025 mM oligodeoxythymidylate(Supertechs 111020A), and 0.25 μCi 3HdTTP (Amersham Life Science,catalog #TRK.424) in 50 μl reaction volume.

Each 45 μl aliquot of the RT reaction mix was mixed with 2 μl (10 units)of one of the DNA polymerases, and 1 μl of either 50 mM MnCl₂ or 50 mMMgCl₂. The solutions were then incubated at 70° C. for 15 minutes.Reactions were stopped by placing them on ice native Taq, sequencinggrade Taq (sTaq), and Tth were from Promega (M166, M203, M210respectively), Tne was purified as described in U.S. Pat. No. 6,001,645incorporated herein by reference. The negative control was performed asdescribed but without addition of any enzyme.

The ³HdTTP incorporation was determined by measuring TCA precipitationcounts as follows. Each RT reaction was TCA precipitated by adding 10 μlcalf thymus DNA (1 mg/ml), 500 μl 10% cold TCA solution, and thenallowed to sit on ice for 10 minutes before it was filtered onto GF/Cfilter (Whatman, 1822024). The filter was washed with 5 ml 5% cold TCAsolution three times, and once with acetone. The filter was dried undera heat lamp, and then counted in a liquid scintillation counter inscintillation fluid (Beckman, 158735). The results (corrected forbackground) are presented in Table 2.

TABLE 2 Reverse Transcriptase Activity ³H-dTTP Incorporation EnzymeMnCl₂ (mM) MgCl₂ (mM) (CPA) native Tvu 1 — 35654 native Tvu — 1 10502Taq 1 — 11110 Taq — 1 70 sTaq⁺ 1 — 9920 sTaq⁺ — 1 192 Tth 1 — 11201 Tth*1 — 19988 Tth* — 1 160 Tne 1 — 14456 Tne — 1 114 *Reaction was done in0.05% Tomah E-18-15 detergent ⁺Sequencing grade Taq

This experiment demonstrated that: 1) the DNA polymerases tested hadhigh RT activity in the presence of Mn²⁺; 2) addition of 0.05% TomahE-18-15 detergent (e.g., U.S. patent application Ser. No. 09/338,174,incorporated herein by reference) increased Tth RT activity by 80% inMn²⁺ buffer; and 3) of the polymerases tested, only Tvu DNA polymerasehas significant reverse transcriptase activity in the presence of Mg²⁺ions. As indicated by the data, the reverse transcriptase activity ofTvu DNA polymerase is approximately 150 times higher than native Taq DNApolymerase, approximately 52 times higher than sequencing-grade Taq DNApolymerase, approximately 66 times higher than Tth DNA polymerase, andapproximately 92 times higher than Tne DNA polymerase in the presence of1 mM MgCl₂.

Example 4 Reverse Transcriptase Activity of Tvu DNA Polymerase TestedOver a Range of Magnesium Concentrations

This example describes the determination of the magnesium ionconcentration at which Tvu DNA polymerase has the highest reversetranscriptase activity. A reverse transcription (RT) reaction mix wasprepared as described in Example 3 above, except that 10 mM KCl (i.e.,instead of 90 mM KCl) was used in the 10×RT buffer. The mix componentsand their concentrations are indicated in Table 3.

TABLE 3 Reverse Transcriptase Reactions Com- ponent Amount 50 mM 1 1.5 22.5 0 0 0 0 0 MgCl₂ (μl) 100 mM 0 0 0 0 1.5 1.75 2 2.5 0 MgCl₂ (μl) 5u/μl 2 2 2 2 2 2 2 2 0 Tvu (μl) RT 45 45 45 45 45 45 45 45 45 reactionmix (μl) Mg²⁺ Concentration in Each Reaction (mM) 1.0 1.5 2.0 2.5 3.03.5 4.0 5.0 0

Each reaction was incubated at 70° C. for 20 minutes. Reactions werestopped by placing them on ice. The ³HdTTP incorporation was determinedby measuring TCA precipitation counts as described in Example 3. Theresults are presented in Table 4 (all values shown were corrected forbackground).

TABLE 4 Reverse Transcriptase Assay MgCl₂ (mM) ³HdTTP Incorporation(CPA) 1.0 14464 1.5 22787 2.0 25427 3.0 32395 3.5 25580 4.0 27472 5.026487

This experiment demonstrates that the reverse transcriptase activity ofTvu DNA polymerase increased at levels from 1 to 3 mM Mg²⁺, was maximumat 3 mM Mg²⁺, and then decreased when the Mg²⁺ concentration wasincreased above 3 mM.

Example 5 Reverse Transcriptase Activity of Tvu DNA Polymerase TestedOver a Range of Manganese Ion Concentrations

This experiments describes the determination of the optimum Mn²⁺concentration for reverse transcriptase activity. A reversetranscription (RT) reaction mix was prepared as described in Example 3,except that Tomah E-18-15 detergent was added to a final concentrationof 0.01%, and Tvu DNA polymerase was added to a final concentration of0.07 units per μl of RT reaction mix. The mix components are indicatedin Table 5.

TABLE 5 Reverse Transcription Reactions Component Amount 25 mM MnCl₂(μl) 0 0 1.2 1.4 1.6 1.8 2.0 10 mM MnCl₂ (μl) 2 2.5 0 0 0 0 0 RTreaction mix (μl) 45 45 45 45 45 45 45 Mn²⁺ Concentration in EachReaction (mM) 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Each reaction was incubated at 74° C. for 20 minutes. Reactions werestopped by placing them on ice. The ³HdTTP incorporation was determinedby measuring TCA precipitation counts as described in Example 3. Theresults are shown in Table 6 (all values shown were corrected forbackground).

TABLE 6 Reverse Transcriptase Activity MnCl₂ (mM) ³HdTTP incorporation(CPA) 0.4 7670 0.5 8258 0.6 9200 0.7 8718 0.8 7600 0.9 7616 1.0 7610

This experiment demonstrates that the reverse transcriptase activity ofTvu DNA polymerase increased as the level of Mn²⁺ in the reactionincreased from 0.4 to 0.6 mM, was maximum at 0.6 mM Mn²⁺, and decreasedwhen Mn²⁺ concentration was increased above 0.6 mM.

Example 6 Tvu and Bst Reverse Transcriptase Activity in Mg²⁺ Buffer

This example compares the reverse transcriptase (RT) activity of Tvu DNApolymerase with that of Bst DNA polymerase (NEB, 275L). In theseexperiments, a RT reaction mix was prepared with the final concentrationof each component of the mix in a reaction: 50 mM Tris-HCl (pH 8.3), 40mM KCl, 0.5 mM dTTP (Promega, U123A), 7 mM MgCl₂, 10 mM DTT, 0.25 mMpolyriboadenylate, 0.025 mM oligodeoxythymidylate (Supertechs,#111020A), and 0.25 Ci ³HdTTP (Amersham, TRK.424) in a 50 μl reaction.

A 45 μl aliquot of the RT reaction mix was mixed with 1.25 units enzyme.The solution was then incubated at 70° C. for 15 minutes for the Tvu DNApolymerase, and 65° C. for 15 minutes for the Bst DNA polymerase. Thereactions were stopped by placing them on ice. The experiment wasrepeated for differing amounts of enzyme. A negative control wasperformed without any enzymes.

The results are presented in Table 7 (all values shown were correctedfor background).

TABLE 7 Reverse Transcriptase Activity Enzyme Units ³HdTTP Incorporation(CPM) Tvu DNA Polymerase at 74° C.   1.25 2054   2.5 2890 5 15786 BstDNA Polymerase at 65° C.   1.25 26374   2.5 34492 5 39602 8 52757

This example demonstrates that Bst DNA polymerase has reversetranscriptase activity in the presence of Mg⁺.

Example 7 Thermostability of Tvu DNA Polymerase

This example was performed to determine the thermostability of Tvu DNApolymerase. Tvu DNA polymerase (0.08 units) was added to 55 μl of DNApolymerase activity assay mix described in Example 2. The solution wasincubated at 70° C. for 10 minutes. The reaction was terminated byplacing the tube on ice. The ³H-dTTP incorporation was determined bymeasuring TCA precipitation counts (See Example 2). The experiment wasrepeated using incubation temperatures of 72, 74, 76, 78, and 80° C. Theresults are presented in Table 8 (all values were corrected forbackground).

TABLE 8 Thermostability Temperature (° C.) ³H-dTTP Incorporation (CPM)70 7458 72 6556 74 3834 76 1202 78 790 80 596

This experiment demonstrates that Tvu DNA polymerase activity decreasesas the temperature increases above 70° C. and that the optimaltemperature for Tvu DNA polymerase activity is about 70° C. or lower.

Example 8 Comparison of Bst Reverse Transcriptase Activity in thePresence of Mg²⁺ or Mn²⁺

In this example, the reverse transcriptase activity of Bst DNApolymerase in reaction mixes comprising either Mg²⁺ or Mn²⁺ wascompared. A reverse transcription (RT) reaction mix was prepared as inExample 3, except that Tomah E-18-15 detergent was added to the mix to afinal concentration of 0.1%. A 45 μl aliquot of the RT reaction mix wasmixed with 1 μl (8 units) enzyme, and 1 μl of either 50 mM MnCl₂ or 100mM MgCl₂, and 3 μl 1% Tomah E-18-15 detergent. The solutions were thenincubated at 65° C. for 20 minutes. Reactions were stopped by placingthem on ice. A negative control was performed as described, with theabsence of any enzyme. The ³HdTTP incorporation was determined bymeasuring TCA precipitation counts as described in Example 3. Theresults are presented in Table 8 (all values shown were corrected forbackground).

TABLE 9 Reverse Transcriptase Activity ³HdTTP Incorporation MgCl₂ (mM)MnCl₂ (mM) (CPM) — 1 69476 2 — 49560

This example demonstrates that Bst DNA polymerase has reversetranscriptase activity in the presence of both Mg²⁺ and Mn²⁺ ions.

Example 9 Tvu and Bst Reverse Transcriptase Activity at High Temperature

This example was performed to determine the optimum temperature for thereverse transcriptase activity of Tvu and Bst DNA polymerase. A 25 μlsolution, containing 2.5 units Tvu or Bst DNA polymerase, 2 mM MgCl₂,and 1×RT reaction mix (See Example 3) was made. The solution wasincubated at 65° C. for 10 minutes. The reaction was then terminated byplacing it on ice. The ³HdTTP incorporation was determined by measuringTCA precipitation counts as described in Example 3. The experiment wasrepeated using incubation temperatures of 68, 70, 72, 74, 76, and 78° C.The results obtained are presented in Table 10 (results were correctedto remove background).

TABLE 10 Reverse Transcriptase Activity at High Temperature Temperature(° C.) ³HdTTP Incorporation (CPM) Tvu DNA Polymerase 65 1756 68 1906 701458 72 1432 74 620 76 560 78 530 Bst DNA Polymerase 65 3356 68 2364 701294 72 1258 74 1298 76 1186 78 1360

This experiment demonstrates that Tvu DNA polymerase reversetranscriptase activity increases as the reaction temperature rises from65° C. to 68° C., is maximum at 68° C., and then decreases attemperatures above 74° C. This suggests that the optimal temperature forthe reverse transcriptase activity of Tvu DNA polymerase isapproximately 68° C. The Bst DNA polymerase reverse transcriptaseactivity was maximum at 65° C., and these data suggest that the optimaltemperature for Bst reverse transcriptase activity is at or below about65° C.

Example 10 Tvu and Bst DNA Polymerase PCR

To demonstrate that Tvu DNA polymerases can be used to perform PCR, thefollowing experiment was performed. A 49 μl solution, containing PCRbuffer, dNTP (Promega U1240), template DNA, primer A, primer B (DNAsdescribed below), and additives (Betaine for Bst, Formamide for Tvu) wasmade. The solution was incubated in a thermocycler at 95° C. for 2minutes. The solution was then cooled to and incubated at 65° C. for 2minutes. During this time, 1 μl of Bst (8 u/μl) or Tvu DNA polymerase (5u/μl) was added to the solution to bring the final concentration of eachcomponent to the following: 10 mM Tris-HCl (pH7.5), 50 mM NaCl, 10 mMMgCl₂, 1.5 mM dNTP, 10 ng template DNA, 1 μM primer A, 1 μM primer B,and 1M Betaine for Bst or 0.5% Formamide for Tvu. The solution wasincubated for 35 cycles (75° C. for 15 seconds, and 65° C. for 2minutes). The final extension reaction was performed at 65° C. for 5minutes. The reaction was then stored at 4° C. Ten μl of the reactionwere then loaded onto a 20% TBE gel (Novex, EC6315). The gel was run at230 volts for 60 minutes and stained with ethidium bromide. A 36 bp bandwas detected for both DNA polymerase reactions. This exampledemonstrates that both Bst and Tvu DNA polymerases are capable ofperforming PCR under the conditions described in this example.

In these experiments, Primer A (Promega, 9078) had the followingsequence: 5′-GACGTCGCATGCTCCT-3′ (SEQ ID NO:7); while Primer B (Promega,9080) had the following sequence: sequence 5′-ACCGAATTCCTCGAGTC-3′ (SEQID NO:8). Template DNA was made by digesting plasmid pGEM-7fz+ (Promega,p225A) with restriction enzymes ApaI and KpnI.

Example 11 Cloning Recombinant Tvu DNA Polymerases Wild-Type and MutantForms

Cloning of Gene Encoding Wild-Type Tvu DNA polymerase

Genomic DNA was isolated from Tvu and used to clone the full-length TvuDNA polymerase into an expression vector. Two mutant recombinant Tvu DNApolymerases were then constructed, both of which have deleted the 5′ to3′ exonuclease-encoding domain.

Genomic DNA was isolated from Tvu by resuspending Tvu cells grownovernight in Luria Broth in TE (10 mM Tris, 1 mM EDTA) and vortexingvigorously. The cell solution was then combined with 0.1 mm glass/zirconbeads and beaten at 5000 rpm for 2 cycles of 20 seconds each. The cellswere then fully dispersed and appeared to be lysed. The liquid wastransferred to a fresh tube and extracted twice with phenol and oncewith chloroform. Each time the aqueous phase was transferred to a cleantube. The aqueous phase was then treated with RNase I and ethanolprecipitated. The DNA was spooled and washed in 70% ethanol beforedrying. The dried DNA pellet was then resuspended in TE to a finalconcentration of 3 μg/μl.

The DNA polymerase domain was amplified from the Tvu genomic DNA by PCR.The following components were combined:

Tvu genomic DNA (predenatured at 98° C., 2 minutes) 1 μl Primer JH47(500 picomoles) 1 μl Primer JH49 (500 picomoles) 1 μl 10X Taq bufferwith 15 mM MgCl₂ (Promega, 5 μl 10 mM dNTPs 1 μl Nanopure water 40 μl The sequence of the degenerate primers used are conserved in DNApolymerases and are listed below:

JH47 TAGAGCGGCCGCGAYCCIAAYYTICARAAYAT (SEQ ID NO: 9) JH49CTGCGGCCGCCTAIIACIAIYTCRTCRTGIAC (SEQ ID NO: 10)

Y indicates a pyrimidine (T or C)

I indicates inosine which anneals with any of the four conventionalbases

R indicates a purine (A or G)

The PCR cycling profile was: 96° C., 1 min (94° C., 15 sec; 32° C., 30sec; 72° C., 1 min)×25 cycles, 72° C. 1 minute. A 600 base pair fragmentwas produced as expected. The PCR product was purified with Wizard PCRPurification System (Promega, A7170) according to manufacturer'sinstructions. Twenty-five nanograms of the fragment was ligated to 50 ngT-vector (Promega, A3600) according to manufacturer's instructions. Fourmicroliters of the ligation was transformed into competent JM109 cells.Clones were selected, digested with the Pvu II restriction enzyme anddemonstrated to contain the 600 base pair PCR product. The product wassequenced by dideoxy sequencing. When the resulting amino acid sequenceencoded by this polynucleotide was compared to the amino acid sequenceof E. coli PoIA and Taq DNA polymerase, it demonstrated about 50%homology to both, indicating that the cloned PCR product originated fromthe DNA polymerase gene of Tvu.

Oligonucleotide 11300 (5′-GCGCGAAGAACGGCTGCAGGC-3′, SEQ ID NO:11) whichis within the 600 bp PCR fragment was labelled with ³³P-ATP using T4polynucleotide kinase and used as a probe for a Southern blot. TheSouthern blot had Tvu genomic DNA digested with one of seven differentrestriction enzymes (BamH I, Acc65 I, Apa I, EcoR I, Hind III, Spe I,Xba I, Xho I) per lane. The prehybridization conditions were 65° C., 1.5hours in 3 ml of 1×SSPE, 10% PEG-8000, 7% SDS, 250 μg/ml denaturedHerring Sperm DNA. Hybridization conditions were the same solution asused for the prehybridization with the addition of the radiolabeledprobe purified on a G-25 column and reaction at 50° C. for four hours.The washes were 15 to 30 minutes each, 200 ml of 0.3×SSC, 0.1% SDS at25° C., repeated, followed by three washes of 200 ml of 0.3×SSC, 0.1%SDS at 50° C. The blot was then exposed to X-OMAT film for 2 days at 22°C. There was one band of about 3 kb detectable in the Hind III digestedlane and one band larger than 10 kb detectable in the Xho I digestedlane.

Tvu genomic DNA was digested with Hind III restriction enzyme and runinto a 0.4% TAE agarose gel. The region near the 3 kb position was cutout of the gel, purified with Wizard PCR Purification System (Promega,A7170). The purified 3 kb fragment was ligated into pZERO-2 (Invitrogen)and transformed into TOP10 cells (LTI). Ninety-six clones were pickedand each grown in 200 ul LB media containing 30 ug/ml kanamycin, shakingovernight at 37° C. The cultures were dot blotted using oligonucleotide11300 described above as the probe and prehybridization andhybridization conditions also described above. The washes were two 150ml washes of 0.5×SSC, 0.1% SDS at 25° C., 15-30 minutes each, followedby three 150 ml washes of 0.5×SSC, 0.1% SDS at 50° C., 15-30 minuteseach. The blot was then exposed to X-OMAT film for two hours anddeveloped. Two colonies produced a strong signal. They were grown andplasmid isolated therefrom. The cloned fragments in the plasmids weresequenced and indicated that the Hind III restriction enzyme site was183 base pairs upstream of the QNIP conserved region indicating aboutone third of the DNA polymerase gene (the C-terminus) was present in theclone.

To clone upstream of the Hind III site in the gene, a second PCRamplification was designed to amplify the region upstream of the HindIII site. Again, a degenerate primer (JH31) was used that containedconserved sequence present in DNA polymerases. The second primer (11299)was chosen from within the previously cloned Hind III fragment of TvuDNA polymerase. The following PCR reaction was assembled:

Tvu genomic DNA 1 μl JH31 primer 400 pmoles 4 μl 11299 primer 50 pmoles5 μl 10 mM dNTPs 1 μl 10X Taq buffer 5 μl 50 mM MgSO₄ 2 μl Taqpolymerase 1 μl Water/enhancer 31 μl 

JH31 TTCAACCIIAACTCIIIIAICAGCT (SEQ ID NO: 12) 11299CGGCTCCGACGGCACGAACG (SEQ ID NO: 13)

The PCR cycling conditions were 96° C., 1 minute (94° C., 15 sec; 37°C., 30 sec; 72° C., 1 minute)×25, 72° C., 1 minute. The PCR reaction wasrun on a 1.2% TBE/agarose gel. The resulting 350 bp band was as expectedand was purified using Wizard PCR Purification System (Promega, A7170).The fragment was ligated into a T-vector and transformed into JM109cells. Positive clones were sequenced. The sequence downstream from theHind III site was identical to the previous clone. The sequence upstreamof the Hind III site encoded amino acids homologous to other DNApolymerases.

New Tvu genomic DNA was isolated as previously described, except thatcells were lysed with proteinase K, in order to obtain DNA that was lesssheared than the present stock. An oligonucleotide (11761) was preparedusing sequence upstream of the Hind III site obtained as describedabove. This oligonucleotide sequence is listed below.

11761 TCAACACCGGGAGCTGCAGCTTGTCA (SEQ ID NO: 14)

Tvu genomic DNA was digested with Hind III or Hind III plus anotherrestriction enzyme (Acc I, BamH I, Bgl II, EcoR I, Spe I, Xba I, Xho I,Xho II) and each digested sample run on a lane of a 0.6% TBE/agarosegel. The DNA in the gel was transferred to a nylon membrane by Southernblot procedure. The 11761 oligonucleotide was end labelled with³³P-gamma-ATP using T4 polynucleotide kinase and purified over a NAP-5column (Pharmacia) according to manufacturer's instructions.Prehybridization, Hybridization, and Wash conditions were as previouslydescribed. The membrane was then exposed to X-OMAT film for several daysand developed. There was a 4 kb band in all of the lanes except for theHind III+EcoR I digest lane in which the band was slightly smaller.These results indicate that there is a Hind III restriction enzyme sitelocated about 4 kb upstream of the Hind III site previously localized tothe coding sequence of Tvu DNA polymerase.

Tvu genomic DNA was digested with Hind III and run into a 0.6%TBE/agarose gel. The agarose at the 4 kb position was cut out of the geland the DNA isolated. The resulting DNA was ligated into pZERO-2(Invitrogen) at the Hind III site and transformed into TOP 10 cells.Clones were screened by dot blot as described above using the 11761radiolabeled oligonucleotide as the probe. A positive clone was grown,the plasmid purified, and the insert containing the remainder the TvuDNA polymerase gene was sequenced.

The two Hind III fragments were cloned in correct order into Litmus 29plasmid (New England Biolabs) and resequenced across fragment junctions.This full length clone of Tvu DNA polymerase in Litmus 29 plasmid isnamed L29b. The resulting open reading frame nucleotide sequence is SEQID NO: 1.

Mutant Tvu DNA Polymerase Construction—T289M

The construction of T289M mutant of Tvu DNA polymerase resulted in aplasmid containing an IPTG-inducible mammalian promoter directingexpression of the Tvu DNA fragment beginning at the nucleotides encodingamino acid 289 of the wild type enzyme, mutated to encode a methionineresidue instead of a threonine, and ending at the termination codon ofthe wild type enzyme.

The JHEX25 vector (Promega) was digested with Nco I and Acc65 Irestriction enzymes and the large linear band isolated from an agarosegel. The L29b vector, described above, was digested with Sgf I and Acc65I restriction enzymes and the 1.8 kb band isolated from an agarose gel.The Sgf I cut site in L29b is located 912 base pairs downstream from thepolymerase start codon and the Acc65 I cut site in L29b is located 69base pairs downstream from the polymerase termination codon.

Oligonucleotides 12144 and 12145 were designed such that when they areannealed to each other an Sgf I overhang exists on one end and an Nco Ioverhang exists on the other end. The ATG within the Nco I site createsthe new, non-native start site for the T289M DNA polymerase. Theoligonucleotides were annealed by combining in a tube 2 pmols/μl of eachin TNE (10 mM Tris, 5 mM NaCl, 1 mM EDTA), placing the tube in a 9600thermocycler and slowly decreasing the temperature from 80° C. to 25° C.over a period of 40 minutes.

The purified Sgf I/Acc65 I fragment of L29b was ligated to 2 pmols ofannealed 12144/12145 oligonucleotides using T4 DNA ligase at roomtemperature for about two hours. Four microliters of the ligationreaction was then transformed into JM109 cells and plated onto LB platescontaining tetracycline. Colonies were screened by isolating plasmid anddigesting with Nco I and Acc65 I restriction enzymes and furtherconfirmed to be correct by dideoxy sequencing across the sequenceencoding the DNA polymerase. The plasmid was named TvuK-25. Thenucleotide sequence encoding the T289M polymerase is shown in FIG. 5,SEQ ID NO: 5. The amino acid sequence of T289M polymerase is shown inFIG. 6, SEQ ID NO: 6.

12144 (SEQ ID NO: 15) CATGGATGAAGGTGAGAAGCCACTGGCCGGGATGGACTTTGCGAT; and12145 (SEQ ID NO: 16) CGCAAAGTCCATCCCGGCCAGTGGCTTCTCACCTTCATC

Mutant Tvu DNA Polymerase Construction—M285

The construction of the M285 mutant of Tvu DNA polymerase resulted in aplasmid containing an IPTG-inducible mammalian promoter directingexpression of the Tvu DNA fragment beginning at the nucleotides encodingthe methionine amino acid at position 285 of the wild type enzyme andending at the termination codon of the wild type enzyme.

The TvuK-25 plasmid described above was digested with Dra I and Sgf Irestriction enzymes. The large linear band was isolated from an agarosegel. Oligonucleotides 12230 and 12231 were designed such that when theyare annealed to each other an Sgf I overhang exists on one end and a DraI overhang exists on the other end. The oligonucleotides were annealedby combining in a tube 2 pmols/μl of each in TNE (10 mM Tris, 5 mM NaCl,1 mM EDTA), placing the tube in a 9600 thermocycler and slowlydecreasing the temperature from 80° C. to 25° C. over a period of 40minutes.

The purified Sgf I/Dra I fragment of TvuK-25 was ligated to 2 pmols ofannealed 12230/12231 oligonucleotides using T4 DNA ligase at roomtemperature for about two hours. Four microliters of the ligationreaction was then transformed into JM109 cells and plated onto LB platescontaining tetracycline. Colonies were screened by isolating plasmid anddigesting with either Dra I or AccB7 I restriction enzymes and furtherconfirmed to be correct by dideoxy sequencing across the sequenceencoding the DNA polymerase.

The nucleotide sequence encoding the M285 polymerase is shown in FIG. 3,SEQ ID NO: 3. The amino acid sequence of M285 polymerase is shown inFIG. 4, SEQ ID NO: 4.

12230 (SEQ ID NO: 17) AAACCATGGCAGTTCAAACCGATGAAGGCGAGAAACCACTGGCTGGGATGGACTTTGCGAT; and 12231 (SEQ ID NO: 18)CGCAAAGTCCATCCCAGCCAGTGGTTTCTCGCCTTCATCGGTTTGAACTG CCATGGTTT

Example 12 Expression and Purification of Recombinant Tvu DNAPolymerases

The recombinant Tvu DNA polymerases, both full-length and mutant, wereexpressed and purified as described herein. For the full-length clone, aliter of Terrific Broth containing 100 ug/ml ampicillin was grown at 37°C. to saturation with E. coli transformed with the vector capable ofexpressing recombinant full-length Tvu DNA polymerase (described inExample 11). The cells were harvested by centrifugation at 9,000 rpm for5 minutes.

For the full-length recombinant Tvu DNA polymerase, 20 g cell paste wascombined with 200 ml of 0.25 M NaCl TEDG (50 mM Tris-HCl at pH 7.3, 1 mMEDTA, 1 mM DTT, and 10% Glycerol) containing 2.5 mM PMSF. The solutionwas sonicated at 100% output three times for two minutes each at 10° C.The solution (40 ml aliquots) was then heat treated at 65° C. for 5minutes and then cooled to 4° C. Then 4 ml of 5% PEI was added to thelysate to precipitate the DNA. The following purification steps wereperformed at 4° C. Centrifugation (12,000 rpm in a Beckman JA18 rotorfor 90 minutes) was used to separate the supernatant from theprecipitate. The supernatant was then collected, and ammonium sulfatewas added to a final saturation of 65% to precipitate the DNApolymerase. Centrifugation (15,000 rpm in a Beckman JA18 rotor for 30minutes) was used to separate the ammonium sulfate precipitate from thesupernatant. The precipitate was collected, suspended in TEDG buffer anddialyzed against TEDG buffer containing 2.5 mM PMSF overnight to removethe ammonium sulfate.

The dialyzed solution was then loaded onto a Heparin-Agarose column (SPL1905-0004) equilibrated with TEDG buffer. After washing the column withTEDG buffer, elution was performed by applying a linear gradient of 0 to0.6 M NaCl TEDG buffer. The fractions were collected, and assayed forDNA polymerase activity as described in Example 2. The presence ofendonucleases was determined by incubating 2 μl of fractions with 1 μglambda DNA (Promega, D150) or pBR322 plasmid DNA in activity assaybuffer for 17 hours at 70° C. Agarose gel analysis of the digest showedno evidence of nuclease contamination. Fractions with DNA polymeraseactivity were pooled. The pooled fractions were dialyzed against TEDGbuffer, then loaded onto a TEDG buffer equilibrated Cibacron Blue column(Sigma, C-1535). After washing the column with 0.05 M NaCl/TEDG buffer,elution was performed with a linear gradient of 0.05 to 0.75 M NaCl/TEDGbuffer. The eluate was collected in fractions, and sample fractions wereassayed for DNA polymerase activity and retested for nucleasecontamination. No such contamination was detected. The fractions withDNA polymerase activity were pooled and Tomah-34 detergent added to afinal concentration of 0.2% (e.g., U.S. patent application Ser. No.09/338,174, incorporated herein by reference). The polymerase solutionwas then dialyzed overnight against the storage buffer (50% glycercol,20 mM Tris, pH 8.0 at 25° C., 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5%Toman-34).

The mutant Tvu DNA polymerases (M285 and T289M) encoded byIPTG-inducible plasmids. For growth of these mutant plasmids, 3 litersof Terrific Broth containing 10 ug/ml tetracycline were seededseparately with 50 ml overnight seedstocks of E. coli containing eithermutant plasmid. The cultures were grown to about A600=1.5 OD shaking at37° C. Then the culture growth temperature was adjusted to 25° C. andIPTG was added to a final concentration of 1 mM. The culture was allowedto grow overnight, shaking at 25° C. and the cells were then harvestedby centrifugation at 9,000 rpm for 5 minutes. The purification procedureis then the same as that described above for the full-length rTvu DNApolymerase.

This experiment demonstrated that the recombinant Tvu DNA polymeraseswere purified to greater than 95% as indicated by a predominant band atabout 97 kD for the full-length polymerase and 66 kD for the mutantpolymerases when compared to Mark 12 size markers (Novex) on a 4-20%Tris-Glycine gel (Novex EC6025).

Example 13 Use of Recombinant Tvu DNA Polymerases in ReverseTranscription Reaction

Reverse transcription activity in the presence of magnesium ions wasmeasured for the full-length and mutant recombinant Tvu DNA polymeraseenzymes purified as described in Example 12.

In these experiments, a reverse transcription (RT) reaction mix wasused. The final concentration of each component in a reaction was: 10 mMTris-HCl (pH 8.3), 50 mM KCl, 0.5 mM dTTP (Promega, U123A), 0.25 mMpolyriboadenylate, 0.025 mM oligodeoxythymidylate (Supertechs 111020A),and 0.25 μCi ³HdTTP (Amersham, TRK.424) in 50 μl reaction volume.

Each 45 μl aliquot of the RT reaction mix was mixed with 2 μl (10 units)or 1 μl (5 units) of one of the DNA polymerases and water to a finalvolume of 50 μl. The solutions were then incubated at 74° C. for 20minutes. Reactions were stopped by placing them on ice. The negativecontrol was performed as described but without addition of any enzyme.

The ³HdTTP incorporation was determined by measuring TCA precipitationcounts as follows. Each RT reaction was TCA precipitated by adding 10 μlcalf thymus DNA (1 mg/ml), 500 μl 10% cold TCA solution, and thenallowed to sit on ice for 10 minutes before it was filtered onto GF/Cfilter (Whatman, 1822024). The filter was washed with 5 ml 5% cold TCAsolution three times, and once with acetone. The filter was dried undera heat lamp, and then counted in a liquid scintillation counter inscintillation fluid (Beckman, 158735). The results (corrected forbackground) are presented in Table 10.

TABLE 10 Reverse Transcription Activity of Recombinant Tvu DNAPolymerase Enzyme Amount of Enzyme cpm Full Length rTvu DNA pol.  5units 12,560 Full Length rTvu DNA pol. 10 units 18,794 M285  5 units13,202 M285 10 units 19,390 T289M  5 units 8,434 T289M 10 units 16,264

The results demonstrate that all recombinant Tvu DNA polymerases testedhave reverse transcriptase activity at 74° C., and 10 units producedmore activity than 5 units as expected.

Example 14 nTvu Reverse Transcriptase Functions in a Single-Step RT-PCRReaction

This experiment describes the use of nTvu DNA polymerase in a singlestep RT-PCR reaction. A 30 μl solution, containing 10 mM Tris-HCl(pH8.3), 50 mM KCl, 0.2 mM dNTP, 0.3 μM primer C (Promega, A109B), 0.3μM primer D (Promega, A110B), 1.5 mM MgCl₂, 5 units Tvu DNA polymerase,5 units Taq DNA polymerase, and 10⁻¹² moles Kanamycin mRNA (Promega,C138A), was prepared on ice. The solution was then incubated at 70° C.for 20 minutes in a thermocycler before the start of PCR: 95° C. for 1minute, followed by 35 cycles (94° C. for 15 seconds, 60° C. for oneminute), and ended with a final extension at 60° C. for 5 minutes.Following this step, the products were stored at 4° C. The experimentwas repeated for 10⁻¹³, 10⁻¹⁴, 10⁻¹¹, 10⁻¹⁶, 10⁻¹⁷, 10⁻¹⁸, 10⁻¹⁹, 10⁻²⁰,10⁻²¹ moles of kanamycin mRNA. The negative control experiment wasperformed without mRNA template. Ten μl of each reaction were loadedonto a 20% TBE gel and treated as in Example 10. A band of correct sizewas detectable in all lanes.

In these experiments, Primer C (Promega, A109B) had the followingsequence 5′-GCC ATT CTC ACC GGA TTC AGT CCG T-3′ (SEQ ID NO:23). PrimerD (Promega, A110B) had the following sequence 5′-AGC CGC CGT CCC GTC AAGTCA G-3′ (SEQ ID NO:24).

This experiment demonstrates that the reverse transcriptase activity ofTvu DNA polymerase is capable of performing RT under the RT-PCRconditions described in this Example and treated as in Example 10. Aband of correct size was detectable in all lanes.

Example 15 Tvu and Bst DNA Polymerases can Act as Reverse Transcriptasesin Reverse Transcriptase Assays

This experiment describes the use of Tvu and Bst DNA polymerases inRT-PCR assays. For these experiments, a 50 μl solution, containing 10 mMTris-HCl (pH8.3), 50 mM KCl, 0.2 mM dNTP, 0.3 μM primer C (Promega,A109B), 0.3 μM primer D (Promega, A110B), 1.5 mM MgCl₂, either 5 unitsTvu, or 5 units Bst DNA polymerase, and 0.5 μg Kanamycin RNA (Promega,C138A), was prepared on ice. A negative control experiment was carriedout without mRNA template. The solution was incubated for 20 minutes ateither 70° C. for Tvu, or 65° C. for Bst. Then, 5 units Taq DNApolymerase were added before the start of PCR, which was carried out at95° C. for 1 minutes, followed by 35 cycles (94° C. for 15 seconds, 60°C. for 1 minute), and ended with a final extension at 60° C. for 5minutes. Following this step, the products were stored at 4° C. Ten μlof each PCR reaction were loaded onto a 20% TBE gel and processed as inExample 10. The PCR product was purified using a Qiaquick PCRpurification kit (Qiagene, 28104). DNA concentration was estimated usingthe READIT DNA quantitation method (See, e.g., U.S. application Ser. No.09/042,287, incorporated herein by reference). Both strands of PCRproducts were sequenced, and were found to be completely complementary,indicating no mutations were introduced during the reaction. Thisexample demonstrates that the reverse transcriptase activity of Tvu andBst DNA polymerases is capable of performing RT function faithfully.

Example 16 Comparison of the Reverse Transcriptase Activity of Tli, andPwo DNA Polymerases in Mg²⁺ or Mn²⁺ Buffer

This example demonstrates the lack of reverse transcriptase activity ofTli and Pwo DNA polymerases in the presence of Mg²⁺ ions. In theseexperiments, a 45 μl aliquot of the RT reaction mix (See Example 3) wasmixed with 2 μl (10 units) enzyme, and 1 μl either 50 mM MnCl₂ or 100 mMMgCl₂, and 1 μl 2.5% Tomah E-18-15 detergent. The solutions were thenincubated at 70° C. for 20 minutes. Reactions were stopped by placingthem on ice. Tli DNA Polymerase (Promega M7101), and Pwo DNA Polymerase(Boehringer Mannheim 1644955) were utilized in these experiments. Thenegative control experiment was performed without any enzymes. The³HdTTP incorporation was determined by measuring TCA precipitationcounts as described in Example 3. The results are presented in Table 11(all values were corrected for background).

TABLE 11 Reverse Transcriptase Activity ³HdTTP Incorporation EnzymeMnCl₂ (mM) MgCl₂ (mM) (CPM) Tli 1 — 867 Tli — 2 3 Pwo 1 0 7145 Pwo — 224

This example demonstrates that the reverse transcriptase activity of Tliand Pwo DNA polymerases is significant in the presence of Mn²+ buffer,but much lower in the presence of Mg²⁺ buffer.

Example 17 RT-PCR using Tvu and Taq DNA Polymerase Mixtures

Multiple mixtures of Tvu and Taq DNA polymerases were used, at multiplepHs, to demonstrate that RT-PCR can be performed in a one-pot reactionin the presence of magnesium and the substantial absence of manganeseions.

Kanamycin mRNA (Promega C1381) was used as the nucleic acid substrate inthe RT-PCR reactions. The reactions were assembled as detailed in thetable below.

Reaction number: 1 2 3 4 5 Reaction mix (μl) 43 43 43 43 43 Water 4 4 44 5 nTaq 1 1 1 1 1 mRNA (0.5 mg/ml) 1 1 1 1 1 nTvu (full-length) 1 0 0 00 rTvu (full-length) 0 1 0 0 0 M285 Tvu 0 0 1 0 0 T289M Tvu 0 0 0 1 0

The Taq and Tvu DNA polymerases were all at a concentration of 5 unitsper microliter. nTaq and nTvu are native enzymes, rTvu is therecombinant enzyme. Reaction 5 is the negative control reaction. One setof reactions was at pH 8.3, another set of reactions was at pH 9.0. Thereaction mixture was: 5 μl 10× buffer (500 mM KCl, 100 mM Tris pH 8.3 or9.0); 5 μl 2 mM dNTP, 1 μl Primer I (Promega, A109B); 1 μl Primer 2(Promega, A110B); 5 μl 125 mM MgCl₂; 26 μl water.

The PCR cycling program used was 70° C. for 20 minutes to allow forreverse transcription, followed by 95° C. for 1 minute, (94° C. for 15seconds, 68° C. for 1 minute)×30; 68° C. for 5 minutes, 4° C. soak. Analiquot of the RT-PCR reaction was then run on a 20% TBE gel andethidium bromide stained to visualize the 300 bp product.

All of the Tvu DNA polymerase enzyme-containing reactions producedrobust RT-PCR product when coupled with nTaq DNA polymerase in the abovereaction. The RT reaction was run at either 70° C. or 78° C. and bothproduced nearly equal amounts of RT-PCR product. Likewise, pH 8.3 and pH9.0 were both efficient and produced nearly equal amounts of RT-PCRproduct. The mutant and full-length Tvu DNA polymerases produced nearlyequal amounts of RT-PCR product.

A 1:10 serial dilution of the mRNA template was performed and thereaction as described above was run using 2 μl of each dilution whenusing a Tvu DNA polymerase. RT-PCR product of 300 bp was detectable evenwhen using an mRNA dilution containing 1 copy in the 2 μl aliquot. M285Tvu produced a RT-PCR product of 300 bp at four logs less serialdilution than did rTvu. The other forms of Tvu were not tested in anRT-PCR reaction in the absence of Taq DNA polymerase. The negativecontrol reactions containing no Tvu DNA polymerase produced nodetectable RT-PCR product.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described compositions and methods of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with particular preferred embodiments, it should beunderstood that the inventions claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art and in fields related thereto are intended tobe within the scope of the following claims.

1-23. (canceled)
 24. A kit for reverse transcription comprising: a) apolymerase selected from Thermoactinomyces vulgaris and Bacillusstearothermophilus polymerase, b) purified RNA, and c) a buffercomprising magnesium ions wherein said buffer is substantially free ofmanganese ions.
 25. The kit of claim 24, further comprising instructionsfor reverse transcription.
 26. The kit of claim 24, wherein said bufferfurther comprises a surfactant.
 27. The kit of claim 24, wherein saidbuffer has a pH of about 6 to
 10. 28-29. (canceled)
 30. The kit of claim24, further comprising at least one additional thermostable DNApolymerase.
 31. A kit for reverse transcription comprising: a) apolymerase selected from Thermoactinomyces vulgaris and Bacillusstearothermophilus polymerase, and b) a buffer comprising magnesium ionswherein said buffer is substantially free of manganese ions. 32-33.(canceled)
 34. The kit of claim 31, further comprising at least oneadditional thermostable DNA polymerase.
 35. The kit of claim 31, furthercomprising instructions for reverse transcription.